scholarly journals Characterization of an Isolate of Citrus Concave Gum-Associated Virus from Apples in China and Development of an RT-RPA Assay for the Rapid Detection of the Virus

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2239
Author(s):  
Zhen Liu ◽  
Zhenfei Dong ◽  
Binhui Zhan ◽  
Shifang Li
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Phylogenetic Analyses ◽  
The United States ◽  
Polymerase Chain Reaction Method ◽  
Large Scale Testing ◽  
Bright Stripe ◽  
Full Length Genome

Apple (Malus domestica) fruits exhibiting bright stripe symptoms were identified in Weihai City, Shandong Province, China. To investigate the virome in the apple samples, the method of high throughput sequencing (HTS) was used to identify the viruses. It was found that the sequence of citrus concave gum-associated virus (CCGaV) was involved in the apple transcriptome dataset. The full-length genome of the CCGaV-Weihai isolate contained two segments, the RNA1 was 6674 nt in size containing a conserved RNA-dependent RNA polymerase (RdRp), and the RNA2 was ambisense, 2706 nt in length, encoding a movement protein (MP) and a coat protein (CP). Sequence alignment and phylogenetic analyses indicated that CCGaV-Weihai was more closely related to CCGaV-H2799 isolated from the apple host in the United States and distantly related to CCGaV-CGW2 from Citrus sinensis in Italy, indicating a possibly geographical and host differentiation of CCGaV isolates. This was the first identification and characterization of CCGaV infecting apples in China. Additionally, a rapid and sensitive reverse transcription recombinase polymerase amplification (RT-RPA) assay technique was established for CCGaV detection in apple plants. The RT-RPA of CCGaV was not affected by other common viruses in apple plants and is about 10-fold more sensitive than the conventional reverse transcription polymerase chain reaction method, which can be used in large-scale testing.

scholarly journals SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 863 ◽  
Author(s):  
Steffen Klein ◽  
Thorsten G. Müller ◽  
Dina Khalid ◽  
Vera Sonntag-Buck ◽  
Anke-Mareil Heuser ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
N Gene ◽  
Extraction Protocol ◽  
Large Scale Testing

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

scholarly journals Virulence Characterization of Puccinia striiformis f. sp. tritici Using a New Set of Yr Single-Gene Line Differentials in the United States in 2010

Plant Disease ◽  
2014 ◽  
Vol 98 (11) ◽  
pp. 1534-1542 ◽  
Author(s):  
Anmin Wan ◽  
Xianming Chen
Keyword(s):  
United States ◽  
Stripe Rust ◽  
Large Scale ◽  
Puccinia Striiformis ◽  
Yellow Rust ◽  
Single Gene ◽  
The United States ◽  
The Past ◽  
History Of

Puccinia striiformis f. sp. tritici causes stripe rust (yellow rust) of wheat and is highly variable in virulence toward wheat with race-specific resistance. During 2010, wheat stripe rust was the most widespread in the recorded history of the United States, resulting in large-scale application of fungicides and substantial yield loss. A new differential set with 18 yellow rust (Yr) single-gene lines was established and used to differentiate races of P. striiformis f. sp. tritici, which were named as race PSTv in distinction from the PST races identified in the past. An octal system was used to describe the virulence and avirulence patterns of the PSTv races. From 348 viable P. striiformis f. sp. tritici isolates recovered from a total of 381 wheat and grass stripe rust samples collected in 24 states, 41 races, named PSTv-1 to PSTv-41, were identified using the new set of 18 Yr single-gene differentials, and their equivalent PST race names were determined on the previous set of 20 wheat cultivar differentials. The frequencies and distributions of the races and their virulences were determined. The five most predominant races were PSTv-37 (34.5%), PSTv-11 (17.5%), PSTv-14 (7.2%), PSTv-36 (5.2%), and PSTv-34 (4.9%). PSTv-37 was distributed throughout the country while PSTv-11 and PSTv-14 were almost restricted to states west of the Rocky Mountains. The races had virulence to 0 to 13 of the 18 Yr genes. Frequencies of virulences toward resistance genes Yr6, Yr7, Yr8, Yr9, Yr17, Yr27, Yr43, Yr44, YrTr1, and YrExp2 were high (67.0 to 93.7%); those to Yr1 (32.8%) and YrTye (31.3%) were moderate; and those to Yr10, Yr24, Yr32, and YrSP were low (3.4 to 5.7%). All of the isolates were avirulent to Yr5 and Yr15.

scholarly journals A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2, the Gene Encoding a Fungal Aminodeoxychorismate Lyase

2007 ◽  
Vol 6 (11) ◽  
pp. 2102-2111 ◽  
Author(s):  
Javier Botet ◽  
Laura Mateos ◽  
José L. Revuelta ◽  
María A. Santos
Keyword(s):  
Large Scale ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Cellular Processes ◽  
Founder Member ◽  
Yeast Genes ◽  
Vacuole Biogenesis ◽  
Reading Frames

ABSTRACT Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.

scholarly journals Characterization of the virome of shallots affected by the shallot mild yellow stripe disease in France

2019 ◽  
Author(s):  
Armelle Marais ◽  
Chantal Faure ◽  
Sébastien Theil ◽  
Thierry Candresse
Keyword(s):  
High Throughput Sequencing ◽  
Phylogenetic Analyses ◽  
New Disease ◽  
Disease Symptoms ◽  
Link Type ◽  
Virus Complex ◽  
Shallot Latent Virus ◽  
Yellow Stripe ◽  
Complete Genomic

AbstractTo elucidate the etiology of a new disease on shallot in France, double-stranded RNAs from asymptomatic and symptomatic shallot plants were analyzed by high-throughput sequencing (HTS). Contigs annotation, molecular characterization and phylogenetic analyses revealed the presence in symptomatic plants of a virus complex consisting of shallot virus X (ShVX, Allexivirus), shallot latent virus (SLV, Carlavirus) and two novel viruses belonging to the genera Carlavirus and Potyvirus, for which the names of shallot virus S (ShVS) and shallot mild yellow stripe associated virus (SMYSaV), are proposed. Complete or near complete genomic sequences were obtained for all these agents, revealing divergent isolates of ShVX and SLV. Trials to fulfill Koch’s postulates were pursued but failed to reproduce the symptoms on inoculated shallots, even though the plants were proved to be infected by the four viruses detected by HTS. Replanting of bulbs from SMYSaV-inoculated shallot plants resulted in infected plants, showing that the virus can perpetuate the infection over seasons. A survey analyzing 351 shallot samples over a four years period strongly suggests an association of SMYSaV with the disease symptoms. An analysis of SMYSaV diversity indicates the existence of two clusters of isolates, one of which is largely predominant in the field over years.The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers MG571549, MH292861, MH389247 to MH389255, and MG910501 to MG910598.

scholarly journals An indirect method to monitor the fraction of people ever infected with COVID-19: An application to the United States

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245845
Author(s):  
Miguel Sánchez-Romero ◽  
Vanessa di Lego ◽  
Alexia Prskawetz ◽  
Bernardo L. Queiroz
Keyword(s):  
New York ◽  
New Jersey ◽  
Large Scale ◽  
Total Population ◽  
Case Fatality ◽  
The United States ◽  
Complementary Approach ◽  
Large Scale Testing ◽  
Asymptomatic Individuals ◽  
Testing Policies

The number of COVID-19 infections is key for accurately monitoring the pandemics. However, due to differential testing policies, asymptomatic individuals and limited large-scale testing availability, it is challenging to detect all cases. Seroprevalence studies aim to address this gap by retrospectively assessing the number of infections, but they can be expensive and time-intensive, limiting their use to specific population subgroups. In this paper, we propose a complementary approach that combines estimated (1) infection fatality rates (IFR) using a Bayesian melding SEIR model with (2) reported case-fatality rates (CFR) in order to indirectly estimate the fraction of people ever infected (from the total population) and detected (from the ever infected). We apply the technique to the U.S. due to their remarkable regional diversity and because they count with almost a quarter of all global confirmed cases and deaths. We obtain that the IFR varies from 1.25% (0.39–2.16%, 90% CI) in Florida, the most aged population, to 0.69% in Utah (0.21–1.30%, 90% CI), the youngest population. By September 8, 2020, we estimate that at least five states have already a fraction of people ever infected between 10% and 20% (New Jersey, New York, Massachussets, Connecticut, and District of Columbia). The state with the highest estimated fraction of people ever infected is New Jersey with 17.3% (10.0, 55.8, 90% CI). Moreover, our results indicate that with a probability of 90 percent the fraction of detected people among the ever infected since the beginning of the epidemic has been less than 50% in 15 out of the 20 states analyzed in this paper. Our approach can be a valuable tool that complements seroprevalence studies and indicates how efficient have testing policies been since the beginning of the outbreak.

Characterization of Point Defect Generation, Migration and Coalescence in Irradiated SiC by Atomistic Simulation

2007 ◽  
Vol 1043 ◽  
Author(s):  
David Farrell ◽  
Noam Bernstein ◽  
Wing Kam Liu
Keyword(s):  
Point Defect ◽  
Nuclear Power ◽  
Large Scale ◽  
The United States ◽  
Ceramic Composites ◽  
Atomic Scale ◽  
Empirical Potential ◽  
Defect Clusters ◽  
Point Defect Clusters

AbstractRenewed interest in nuclear power in the United States has prompted investigations into new reactor designs, resulting in a need to gain a greater understanding of the properties of the materials which are proposed for use in next generation nuclear reactors. This presentation will focus on preliminary results of large-scale empirical potential atomistic studies into the generation of point defect clusters in 3C SiC by particle irradiation and the evolution from point defect clusters to ‘voids’ on the atomic scale. Our working definition of ‘void’ will be explained in the context of small length-scale simulations. The determination of interstitial and vacancy diffusivities for the empirical potential employed and its impact on defect coalescence will be discussed. The characterization of initial damage states for given irradiation conditions will be presented and compared to previous work on ceramics and ceramic-composites.

scholarly journals Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (10) ◽  
pp. 5996-6004 ◽  
Author(s):  
Jan Vinjé ◽  
Sjon J. G. Oudejans ◽  
Jill R. Stewart ◽  
Mark D. Sobsey ◽  
Sharon C. Long
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Phylogenetic Analyses ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Reverse Line Blot ◽  
Source Tracking ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Male Specific

ABSTRACT In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qβ, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

scholarly journals Biologic, Antigenic, and Full-Length Genomic Characterization of a Bovine-Like Coronavirus Isolated from a Giraffe

2007 ◽  
Vol 81 (10) ◽  
pp. 4981-4990 ◽  
Author(s):  
Mustafa Hasoksuz ◽  
Konstantin Alekseev ◽  
Anastasia Vlasova ◽  
Xinsheng Zhang ◽  
David Spiro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genetic Relatedness ◽  
Phylogenetic Analyses ◽  
Point Mutations ◽  
The United States ◽  
Interspecies Transmission ◽  
Adaptive Mutations ◽  
Severe Diarrhea ◽  
Wild Ruminants

ABSTRACT Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.

scholarly journals Noncoding-RNA-Mediated Regulation in Response to Macronutrient Stress in Plants

2021 ◽  
Vol 22 (20) ◽  
pp. 11205
Author(s):  
Ziwei Li ◽  
Peng Tian ◽  
Tengbo Huang ◽  
Jianzi Huang
Keyword(s):  
Noncoding Rna ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Theoretical Study ◽  
Functional Characterization ◽  
Molecular Networks ◽  
Plant Responses ◽  
Crop Breeding ◽  
Target Module

Macronutrient elements including nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S) are required in relatively large and steady amounts for plant growth and development. Deficient or excessive supply of macronutrients from external environments may trigger a series of plant responses at phenotypic and molecular levels during the entire life cycle. Among the intertwined molecular networks underlying plant responses to macronutrient stress, noncoding RNAs (ncRNAs), mainly microRNAs (miRNAs) and long ncRNAs (lncRNAs), may serve as pivotal regulators for the coordination between nutrient supply and plant demand, while the responsive ncRNA-target module and the interactive mechanism vary among elements and species. Towards a comprehensive identification and functional characterization of nutrient-responsive ncRNAs and their downstream molecules, high-throughput sequencing has produced massive omics data for comparative expression profiling as a first step. In this review, we highlight the recent findings of ncRNA-mediated regulation in response to macronutrient stress, with special emphasis on the large-scale sequencing efforts for screening out candidate nutrient-responsive ncRNAs in plants, and discuss potential improvements in theoretical study to provide better guidance for crop breeding practices.

Sign in / Sign up

Export Citation Format

Share Document