scholarly journals Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Mingmin Zhao ◽  
Beatriz García ◽  
Araiz Gallo ◽  
Ioannis E. Tzanetakis ◽  
Carmen Simón-Mateo ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Rna Virus ◽  
Infectious Clone ◽  
Gibson Assembly ◽  
Infectious Clones ◽  
Universal Approach ◽  
One Step ◽  
Step Generation

AbstractAn unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.

scholarly journals Development and characterization of infectious clones of two strains of Usutu virus

2020 ◽  
Author(s):  
Tyler Bates ◽  
Christina Chuong ◽  
Seth A. Hawks ◽  
Pallavi Rai ◽  
Rebecca M. Salgado ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Insect Cells ◽  
Rna Virus ◽  
Infectious Clone ◽  
Bird Species ◽  
Infection Transmission ◽  
Usutu Virus ◽  
Infectious Clones ◽  
Positive Sense

AbstractUsutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. In this report, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in both mammalian and insect cells. Additionally, we observed similar levels of replication and pathogenesis in two mouse models. This reverse genetics system will aid the scientific community in studying and developing USUV infection, transmission, diagnostics, and vaccines.

scholarly journals First report of apple rubbery wood virus 1 in apple in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Guojun Hu ◽  
Yafeng Dong ◽  
Zunping Zhang ◽  
Xudong Fan ◽  
Fang Ren ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Rna Virus ◽  
Rt Pcr ◽  
Apple Rootstocks ◽  
Apple Trees ◽  
Illumina Hiseq ◽  
First Report ◽  
Sequence Contigs ◽  
Apple Stem

More than 30 viral and subviral pathogens infect apple (Malus domestica, an important fruit crop in China) trees and rootstocks, posing a threat to its production. With advances in diagnostic technologies, new viruses including apple rubbery wood virus 1 (ARWV-1), apple rubbery wood virus 2 (ARWV-2), apple luteovirus 1 (ALV), and citrus virus A (CiVA) have been detected (Beatriz et al. 2018; Rott et al. 2018; Hu et al. 2021). ARWV-1 (family Phenuiviridae) is a negative-sense single-stranded RNA virus with three RNA segments (large [L], medium [M], and small [S]). It causes apple rubbery wood disease (Rott et al. 2018) and is found in apple rootstocks, causing leaf yellowing and mottle symptoms in Korea (Lim et al. 2018). To determine virus prevalence in apple trees in China, 200 apple leaf and shoot samples were collected from orchards in Hebei (n = 26), Liaoning (40), Shandong (100), Yunnan (25), and Shanxi (4), and Inner Mongolia (5) in 2020. Total RNA was extracted from the shoot phloem or leaf (Hu et al., 2015) and subjected to reverse transcription (RT)-PCR to detect apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple necrotic mosaic virus (ApNMV), apple scar skin viroid (ASSVd), ARWV-2, ARWV-1, ALV, and CiVA, using primers specific to respective viruses (Supplementary Table 1). The prevalence of ACLSV, ASPV, ASGV, ApNMV, ASSVd, ARWV-2, ARWV-1, ALV and CiVA was found to be 75.5%, 85.5%, 86.0%, 43.0%, 4.0%, 48.5%, 10.5%, 0% and 0%, respectively (Supplementary Table 2). Among the 21 positive samples for ARWV-1, three, five and 13 samples were from Hebei, Liaoning, and Shandong, respectively. Five ARWV-1-positive samples (cultivars Xinhongjiangjun, Xiangfu-1, Xiangfu-2 and Tianhong) showed leaf mosaic symptoms. To confirm ARWV-1 by RT-PCR, amplicons from Xiangfu-1 and Tianhong were cloned into the pMD18-T vector (Takara, Dalian, China), and three clones of each sample were sequenced. BLASTn analyses demonstrated that the sequences (accession nos. MW507810–MW507811) shared 96.9%–98.9% identity with ARWV-1 sequences (MH714536, MF062127, and MF062138) in GenBank. An lncRNA library was prepared for high-throughput sequencing (HTS) with the Illumina HiSeq platform using Xiangfu-1 RNA. A total of 71,613,294 reads were obtained. De novo assembly of the reads revealed 135 viral sequence contigs of ACLSV, ASGV, ASPV, ApNMV, ARWV-1, and ARWV-2. The sequences of contig-100_88981 (302 nt) and contig-100_25701 (834 nt) (accession nos. MW507821 and MW507820) matched those of segment S from ARWV-1, whereas the sequences of contig-100_6542 (1,660 nt) and contig-100_27 (7,364 nt) (accession nos. MW507819 and MW507818) matched those of segments M and L, respectively. To confirm the HTS results, fragments of segments L (744 bp), M (747 bp), and S (554 bp) from Xiangfu-1 and Tianhong were amplified (Supplementary Table 1) and sequenced. The sequences (accession nos. MW507812–MW507817) showed 94.8%–99.9% nucleotide identity with the corresponding segments of ARWV-1. Co-infection of ARWV-1 with ApNMV and/or ARWV-2 was confirmed in 17/21 ARWV-1-positive samples. The prevalence of ARWV-1/ApNMV, ARWV-1/ARWV-2, and ARWV-1/ApNMV/ARWV-2 infections was 61.9%, 71.4%, and 52.4%, respectively. To our knowledge, this is the first report of ARWV-1 infecting apple trees in China. Further research is needed to determine whether and how ARWV-1 affects apple yield and quality.

scholarly journals Differential Gene Expression Between Polymorphic Zooids of the Marine Bryozoan Bugulina stolonifera

2020 ◽  
Vol 10 (10) ◽  
pp. 3843-3857
Author(s):  
Kira A. Treibergs ◽  
Gonzalo Giribet
Keyword(s):  
Gene Expression ◽  
Division Of Labor ◽  
Differential Gene Expression ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Cdna Libraries ◽  
Living Species ◽  
Differential Gene

Bryozoans are a diverse phylum of marine and freshwater colonial invertebrates containing approximately 6,300 described living species. Bryozoans grow by budding new physiologically connected colony members (zooids) from a founding individual that forms from a metamorphosed larva. In some species these zooids come in different shapes and sizes and are specialized to serve different tasks within the colony. A complex interaction of genotype, environment, and developmental pathway shapes zooid fate, however, the specific mechanisms underlying the establishment of this division of labor remain unknown. Here, the first characterization of differential gene expression between polymorphic zooids of a bryozoan colony is presented. The development of different zooid types of lab-cultured Bugulina stolonifera colonies including feeding autozooids, avicularia (derived non-feeding zooids that are homologous to feeding autozooids but shaped like a bird’s beak), and rhizoids (a branching network of non-feeding anchoring zooids) was explored using RNA sequencing, de novo transcriptome assembly, and differential gene expression analyses. High throughput sequencing of cDNA libraries yielded an average of 14.9 ± 1.3 (SE) million high-quality paired-end reads per sample. Data for the first de novo transcriptome assemblies of B. stolonifera and the first characterization of genes involved in the formation and maintenance of zooid types within a bryozoan colony are presented. In a comparison between autozooid and avicularium tissues, 1,097 significant differentially expressed genes were uncovered. This work provides a much-needed foundation for understanding the mechanisms involved in the development of polymorphic zooids and the establishment of division of labor in bryozoans.

scholarly journals Rapid Construction of a Replication-Competent Infectious Clone of Human Adenovirus Type 14 by Gibson Assembly

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 568 ◽  
Author(s):  
Haibin Pan ◽  
Yuqian Yan ◽  
Jing Zhang ◽  
Shan Zhao ◽  
Liqiang Feng ◽  
...  
Keyword(s):  
Infectious Clone ◽  
A549 Cells ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Enzyme Analysis ◽  
Gibson Assembly ◽  
Assembly Method ◽  
Rapid Construction ◽  
One Step ◽  
Genomic Variant

In 1955, Human adenovirus type 14 (HAdV-B14p) was firstly identified in a military trainee diagnosed as acute respiratory disease (ARD) in the Netherlands. Fifty years later, a genomic variant, HAdV-B14p1, re-emerged in the U.S. and caused large and fatal ARD outbreaks. Subsequently, more and more ARD outbreaks occurred in Canada, the UK, Ireland, and China, in both military and civil settings. To generate a tool for the efficient characterization of this new genomic variant, a full-length infectious genomic clone of HAdV-B14 was successfully constructed using one-step Gibson Assembly method in this study. Firstly, the full genome of HAdV-B14p1 strain GZ01, the first HAdV-B14 isolate in China, was assembled into pBR322 plasmid by Gibson Assembly. The pBRAdV14 plasmid, generated by Gibson Assembly, was analyzed and verified by PCR, restriction enzymes digestion and the sequencing. Secondly, viruses were rescued from pBRAdV14-transfected A549 cells. The integrity of the rescued viruses was identified by restriction enzyme analysis. The complete sequence of the infectious clone was further sequenced. No mutation was found in the infectious clone during the construction when compared with the parental virus and pBR322 sequences. The direct immunofluorescence assay indicated the expression of the hexon protein. Finally, typical virions were observed; the one-step growth curves further showed that the DNA replication and viral reproduction efficiency of pBRAd14 derived viruses was similar with that of wild-type HAdV-B14 strain. The successful construction of the replication-competent infectious clone of pBRAdV14 facilitates the development of vaccine and antiviral drugs against HAdV-B14, as well as provides a novel strategy for rapid construction of infectious viral clones for other large-genome DNA viruses.

scholarly journals Anvi’o: an advanced analysis and visualization platform for ‘omics data

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1319 ◽  
Author(s):  
A. Murat Eren ◽  
Özcan C. Esen ◽  
Christopher Quince ◽  
Joseph H. Vineis ◽  
Hilary G. Morrison ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Complex Data ◽  
Omics Data ◽  
Multiple Sources ◽  
Genomic Changes ◽  
Naturally Occurring ◽  
Multiple Dimensions ◽  
Advanced Analysis

Advances in high-throughput sequencing and ‘omics technologies are revolutionizing studies of naturally occurring microbial communities. Comprehensive investigations of microbial lifestyles require the ability to interactively organize and visualize genetic information and to incorporate subtle differences that enable greater resolution of complex data. Here we introduce anvi’o, an advanced analysis and visualization platform that offers automated and human-guided characterization of microbial genomes in metagenomic assemblies, with interactive interfaces that can link ‘omics data from multiple sources into a single, intuitive display. Its extensible visualization approach distills multiple dimensions of information about each contig, offering a dynamic and unified work environment for data exploration, manipulation, and reporting. Using anvi’o, we re-analyzed publicly available datasets and explored temporal genomic changes within naturally occurring microbial populations throughde novocharacterization of single nucleotide variations, and linked cultivar and single-cell genomes with metagenomic and metatranscriptomic data. Anvi’o is an open-source platform that empowers researchers without extensive bioinformatics skills to perform and communicate in-depth analyses on large ‘omics datasets.

scholarly journals Identification and Characterization of a Novel Emaravirus From Grapevine Showing Chlorotic Mottling Symptoms

2021 ◽  
Vol 12 ◽  
Author(s):  
Xudong Fan ◽  
Chen Li ◽  
Zunping Zhang ◽  
Fang Ren ◽  
Guojun Hu ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
High Throughput Sequencing ◽  
Rna Virus ◽  
Reading Frame ◽  
A Genome ◽  
The Family ◽  
Pcr Products ◽  
Chlorotic Mottling

A novel negative-sense, single-stranded (ss) RNA virus was identified in a “Shennong Jinhuanghou” (SJ) grapevine showing severe chlorotic mottling symptoms by integrating high-throughput sequencing (HTS) and conventional Sanger sequencing of reverse transcription polymerase chain reaction (RT-PCR) products. The virus was provisionally named as “grapevine emaravirus A” (GEVA). GEVA had a genome comprising five genomic RNA segments, each containing a single open reading frame on the viral complementary strand and two untranslated regions with complementary 13- nt stretches at the 5′ and 3′ terminal ends. RNA1 (7,090 nt), RNA2 (2,097 nt), RNA3 (1,615 nt), and RNA4 (1,640 nt) encoded putative proteins P1–P4 that, based on their conserved motifs, were identified as the RNA-dependent RNA polymerase, glycoprotein, nucleocapsid protein, and movement protein, respectively. However, the functional role of protein P5 encoded by RNA5 (1,308 nt) could not be determined. Phylogenetic trees constructed based on amino acids of P1 to P4, allocated GEVA in clade I, together with other species-related emaraviruses. These data support the proposal that GEVA is a representative member of a novel species in the genus Emaravirus of the family Fimoviridae. Moreover, when GEVA was graft-transmitted to SJ and “Beta” grapevines, all grafted plants showed the same symptoms, similar to those observed in the source of the inoculum. This is the first report to our knowledge of an emaravirus infecting grapevine and its possible association with chlorotic mottling symptoms.

scholarly journals De Novo Assembly and Transcriptome Characterization of Canine Retina Using High-Throughput Sequencing

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Bhaskar Reddy ◽  
Amrutlal K. Patel ◽  
Krishna M. Singh ◽  
Deepak B. Patil ◽  
Pinesh V. Parikh ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Open Reading Frames ◽  
Dinucleotide Repeats ◽  
Kegg Pathways ◽  
Ion Torrent Pgm ◽  
Retinal Tissue ◽  
Retinol Metabolism ◽  
And Function

We performed transcriptome sequencing of canine retinal tissue by 454 GS-FLX and Ion Torrent PGM platforms. RNA-Seq analysis by CLC Genomics Workbench mapped expression of 10,360 genes. Gene ontology analysis of retinal transcriptome revealed abundance of transcripts known to be involved in vision associated processes. The de novo assembly of the sequences using CAP3 generated 29,683 contigs with mean length of 560.9 and N50 of 619 bases. Further analysis of contigs predicted 3,827 full-length cDNAs and 29,481 (99%) open reading frames (ORFs). In addition, 3,782 contigs were assigned to 316 KEGG pathways which included melanogenesis, phototransduction, and retinol metabolism with 33, 15, and 11 contigs, respectively. Among the identified microsatellites, dinucleotide repeats were 68.84%, followed by trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides in proportions of 25.76, 9.40, 2.52, and 0.96%, respectively. This study will serve as a valuable resource for understanding the biology and function of canine retina.

Identification of the etiological agent of tilapia disease in the Lake of Galillee

2013 ◽  
Author(s):  
Eran Bacharach ◽  
W. Ian Lipkin ◽  
Avigdor Eldar
Keyword(s):  
High Throughput Sequencing ◽  
Rna Virus ◽  
Epidemiological Studies ◽  
Etiological Agent ◽  
Cdna Libraries ◽  
Farmed Fish ◽  
Screening And Surveillance ◽  
Diseased Fish ◽  
Low Passage

Background to the topic. Tilapines serve as the second most important group of farmed fish worldwide. Massive mortality of wild and cultured tilapia has been observed recently in Israel but the pathogen of this disease has not been identified. We proposed to identify the agent responsible for disease.  Major conclusions, solutions, achievements. We characterized the lesions in diseased fish and found that the brain was one of the affected organs. We found conditions to isolate from brains of diseased fish the etiological agent of the tilapia disease and to propagate it in cell culture. This led to the identification of the pathogen as a novel RNA virus, which we named Tilapia Lake Virus (TiLV). Electron microscopy of TiLV revealed virion-like particles and ether/chloroform-sensitivity assays demonstrated that TiLV is enveloped. Low passage TiLV, injected intra-peritoneally to tilapia, induced a disease with over 80% mortality. Cohabitation of healthy with diseased fish demonstrated that the disease is contagious, and that mortalities occur within few days. Fish surviving initial mortality were immune to further TiLV infections, suggesting the mounting of protective immune response. Screening cDNA libraries and high throughput sequencing determined the sequence of TiLV genome. This demonstrated that TiLV is indeed a novel virus and allowed the design of a PCRbased diagnostic test.  Implications, both scientific and agricultural. The characterization of a novel, emerging RNA virus that imposes major threat to the tilapia industry, enables the specific identification of the virus in tilapines. This allows prompt screening and surveillance of TiLV, epidemiological studies, and disease containment. This also potentially opens the way for the development of vaccines against TiLV.

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