scholarly journals Revolution in Evolution of Human “Anellome”

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 50
Author(s):  
Joanna Kaczorowska ◽  
Mila Sparreboom ◽  
Martin Deijs ◽  
Maarten F. Jebbink ◽  
Lia van der Hoek
Keyword(s):  
Genome Sequencing ◽  
Sanger Sequencing ◽  
Rolling Circle Amplification ◽  
Serum Samples ◽  
Rolling Circle ◽  
The Family ◽  
Full Length Genome ◽  
Viral Sequences ◽  
Hiv 1 ◽  
Repeating Patterns

Torque teno viruses (TTVs), the most well-known members of the family Anelloviridae, are not associated with any disease. They are one of the most abundant and divergent entities in the viral world; however, the cause of their variability is not currently known. In this study, a set of longitudinally collected serum samples from two HIV-1 infected and two non-infected persons was analyzed for the presence of TTVs and other Anelloviridae using a genera-specific quantitative PCR. The samples positive for TTVs were selected for the quantitative heteroduplex tracking assay (QHTA), which showed repeating patterns of TTV genotypes. Sanger sequencing of the partial viral sequences revealed that the same strains of TTVs are most probably disappearing and returning in different stages of life, with scarcely any new introductions. The partial sequences were grouped into phylogenetic genogroups, and samples representing each of these genogroups were selected for full-length genome sequencing using an in-house optimized rolling circle amplification (RCA)–Illumina protocol.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

scholarly journals HIV-1 Unique Recombinant Forms Identified in Slovenia and Their Characterization by Near Full-Length Genome Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 63
Author(s):  
Lunar ◽  
Mlakar ◽  
Zorec ◽  
Poljak
Keyword(s):  
Genome Sequencing ◽  
Recombination Event ◽  
Patient Management ◽  
Full Length ◽  
Base Pairs ◽  
Comprehensive Collection ◽  
Subtype B ◽  
Full Length Genome ◽  
Hiv 1 ◽  
Hiv Diversity

Surveillance of HIV circulating recombinant forms (CRFs) is important because HIV diversity can affect various aspects of HIV infection from prevention to diagnosis and patient management. A comprehensive collection of pol sequences obtained from individuals diagnosed with HIV-1 from 2000 to 2016 in Slovenia was subtyped to identify possible unique recombinant forms (URFs). Selected samples were subjected to near full-length genome (NFLG) sequencing and detailed recombination analyses. Discordant subtyping results were observed for 68/387 (17.6%) sequences and 20 sequences were identified as the most probable URFs and selected for NFLG characterization. Further, 11 NFLGs and two sequences of >7000 base pairs were obtained. Seven sequences were identified as “pure” subtypes or already characterized CRFs: subtype B (n = 5), sub-subtype A6 (n = 1), and CRF01_AE (n = 1). The remaining six sequences were determined to be URFs; four displayed a single recombination event and two exhibited a complex recombination pattern involving several subtypes or CRFs. Finally, three HIV strains were recognized as having epidemic potential and could be further characterized as new CRFs. Our study shows that the identification of new CRFs is possible, even in countries where HIV diversity is considered limited, emphasizing the importance of the surveillance of HIV recombinant forms.

Identification of Extrachromosomal Circular DNA in Hop via Rolling Circle Amplification

2016 ◽  
Vol 148 (2-3) ◽  
pp. 237-240 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
David H. Gent ◽  
Robert R. Martin
Keyword(s):  
Electron Microscopy ◽  
Sequence Analysis ◽  
Higher Plants ◽  
Rolling Circle Amplification ◽  
Small Region ◽  
Extrachromosomal Dna ◽  
Circular Dna ◽  
Rolling Circle ◽  
Dna Molecule ◽  
The Family

During a survey for new viruses affecting hop plants, a circular DNA molecule was identified via rolling circle amplification (RCA) and later characterized. A small region of the 5.7-kb long molecule aligned with a microsatellite region in the Humulus lupulus genome, and no coding sequence was identified. Sequence analysis and literature review suggest that the small DNA molecule is an extranuclear DNA element, specifically, an extrachromosomal circular DNA (eccDNA), and its presence was confirmed by electron microscopy. This work is the first report of eccDNAs in the family Cannabaceae. Additionally, this work highlights the advantages of using RCA to study extrachromosomal DNA in higher plants.

scholarly journals Causative Role of Grapevine Red Blotch Virus in Red Blotch Disease

2018 ◽  
Vol 108 (7) ◽  
pp. 902-909 ◽  
Author(s):  
Luz Marcela Yepes ◽  
Elizabeth Cieniewicz ◽  
Björn Krenz ◽  
Heather McLane ◽  
Jeremy R. Thompson ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Systemic Infection ◽  
Causative Role ◽  
Coding Region ◽  
Protein Coding ◽  
Rolling Circle ◽  
Reverse Transcription Pcr ◽  
Leaf Chlorosis ◽  
The Family

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch’s postulates and revealed the causative role of GRBV in red blotch disease.

scholarly journals Blueberry fruit drop-associated virus: A New Member of the Family Caulimoviridae Isolated From Blueberry Exhibiting Fruit-Drop Symptoms

Plant Disease ◽  
2016 ◽  
Vol 100 (11) ◽  
pp. 2211-2214 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
Robert R. Martin
Keyword(s):  
Rolling Circle Amplification ◽  
The United States ◽  
Washington State ◽  
Cloning And Sequencing ◽  
Rolling Circle ◽  
Reading Frame ◽  
Fruit Drop ◽  
Perfect Correlation ◽  
The Family ◽  
Single Field

This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from ‘Bluecrop’ blueberry plants exhibiting fruit-drop symptoms and named Blueberry fruit drop-associated virus (BFDaV). Blueberry fruit drop disease was first detected in blueberry plants in British Columbia, Canada in the late 1990s, and in a single field in northern Washington state in the United States in 2012. Infected bushes abort nearly 100% of their fruit about three weeks prior to harvest, when the berries are about 3 to 5 mm in diameter. At harvest, the affected plants appear taller than healthy ones as there is no fruit weighing down the branches. The virus was amplified from diseased material using rolling circle amplification, followed by enzyme digestion, cloning, and sequencing. The full genome of BFDaV is 9,850 bp in length and contains a single open reading frame, encoding for a polyprotein, and a large noncoding region. Based on the genome size and organization and phylogenetics, BFDaV is proposed as a new and the largest member of family Caulimoviridae. Finally, in mapping part of a field with fruit-drop symptoms, there was a nearly perfect correlation between the presence of the virus and fruit-drop symptoms.

scholarly journals A9 A method to obtain full-length HIV proviral sequences and their sites of integration

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
Sean C Patro ◽  
Xiaolin Wu ◽  
Shuang Guo ◽  
Michael J Bale ◽  
Ann Wiegand ◽  
...  
Keyword(s):  
T Cells ◽  
Genome Sequencing ◽  
Cd4 T Cells ◽  
Sanger Sequencing ◽  
Integration Site ◽  
Clonal Expansion ◽  
Full Length ◽  
Single Genome ◽  
Integration Sites ◽  
Hiv 1

Abstract Accurate definition of the HIV-1 reservoir on antiretroviral therapy (ART) is of paramount importance to the development of curative strategies. Much of this reservoir is derived from clonal expansion of latently infected CD4+ T cells. Methods used to characterize the reservoir include near full-length single-genome sequencing (NFL-SGS) and integration site analysis (ISA). However, current technologies do not link the intact proviruses detected by NFL-SGS to their sites of integration. Therefore, we developed a method to obtain both near full-length single-proviral sequences and their sites of integration. We call our method full-length integrated proviral single-genome sequencing (FLIP-SGS). Genomic DNA from ACH2 and CEM cells mixed at 1:1,000, or patient samples were diluted to a single proviral endpoint. An in-house, optimized whole genome amplification (WGA) method was performed on wells at the endpoint, generating multiple copies of all DNA molecules within each well. The number of proviral copies after WGA was determined by droplet digital PCR targeting the long terminal region (LTR). Forty per cent of each WGA reaction was used to obtain the provirus–host integration sites with ISA (linker ligation, nested PCR, and Illumina sequencing). The remaining fraction was used to amplify the full-length proviruses in four overlapping fragments (LTR-pol, gag-int, int-env, and env-LTR) for Sanger sequencing. WGA performed on the endpoint-diluted ACH2:CEM DNA amplified single-copy HIV-1 proviral templates greater than 500-fold, making it possible to obtain unique integration sites from single proviruses in ACH2 cells, including one that was previously reported (in the NT5C3A gene on chromosome 7) and two that were not previously reported (in the EIF4ENIF1 gene of chromosome 22 and an unknown region of chromosome 6). Near full-length PCR amplification and Sanger sequencing was performed on proviruses integrated in the NT5C3A gene. FLIP-SGS was applied to peripheral blood mononuclear cells from one HIV-1 infected donor with viremia suppressed on ART and yielded integration sites of four genomes that appear to contain large internal deletions. We report a method for near full-length HIV-1 single-genome sequencing combined with host integration site detection that we call FLIP-SGS. This assay will further define clonal expansion of infected CD4+ T cells as a mechanism that maintains the HIV-1 reservoir and as the source of identical sequences observed during therapy and rebound, rather than from ongoing replication.

Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography

2020 ◽  
Vol 166 ◽  
pp. 112442
Author(s):  
Ruben R.G. Soares ◽  
João C. Varela ◽  
Ujjwal Neogi ◽  
Sibel Ciftci ◽  
Manickam Ashokkumar ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Hiv 1

scholarly journals Detection of the prototype of a potential novel genus in the family Papillomaviridae in association with canine epidermodysplasia verruciformis

2006 ◽  
Vol 87 (12) ◽  
pp. 3551-3557 ◽  
Author(s):  
Kurt Tobler ◽  
Claude Favrot ◽  
Gilles Nespeca ◽  
Mathias Ackermann
Keyword(s):  
Rolling Circle Amplification ◽  
Malignant Lesion ◽  
Human Papillomaviruses ◽  
Epidermodysplasia Verruciformis ◽  
The Novel ◽  
Novel Genus ◽  
Rolling Circle ◽  
The Family ◽  
L1 And L2 ◽  
Flat Warts

Epidermodysplasia verruciformis (EV) is a rare human genetic predisposition to develop flat warts, some of which subsequently undergo cancer transformation. Some human papillomaviruses (HPVs), i.e. HPV 5 and 8, have been associated with cancer development as a sequela of EV. As similar diseases have been observed in dogs, it was hypothesized that unknown canine papillomaviruses (CPVs) may exist and that they may be present in cases of canine EV. Consequently, DNA was extracted from a malignant lesion of a dog with EV and circular DNA was amplified by multiple-primed rolling-circle amplification (RCA). Indeed, sequence determination and analysis of the RCA-amplified and cloned DNA from a malignant canine EV lesion resulted in the detection and primary description of a third CPV (CPV3). Typical papillomavirus genes were identified, with deduced amino acid similarities ranging from 20 to 57 % for E1, E2, E6, E7, L1 and L2, respectively. According to the sequence of the L1 gene, which is used for papillomavirus classification, the new isolate meets the majority of criteria needed to declare detection of a novel genus among the papillomaviruses. Thus, CPV3 may represent the prototype of this novel genus. As the novel virus was found in a dog in association with lesions reminiscent of human EV, it should be interesting to test in the future whether this condition can be reproduced in experimental animals. If such were the case, a new model for EV could be established.

Sign in / Sign up

Export Citation Format

Share Document