scholarly journals Viral population analysis and minority-variant detection using short read next-generation sequencing

2013 ◽  
Vol 368 (1614) ◽  
pp. 20120205 ◽  
Author(s):  
Simon J. Watson ◽  
Matthijs R. A. Welkers ◽  
Daniel P. Depledge ◽  
Eve Coulter ◽  
Judith M. Breuer ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Accurate Determination ◽  
Virus Genome ◽  
Viral Population ◽  
Short Read ◽  
Population Turnover ◽  
Variant Detection ◽  
Sequencing Platforms ◽  
Minority Variant

RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline ( http://sourceforge.net/projects/quasr ) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro .

scholarly journals External Quality Assessment for the Determination of Diphtheria Antitoxin in Human Serum

2010 ◽  
Vol 17 (8) ◽  
pp. 1282-1290 ◽  
Author(s):  
Paolo Di Giovine ◽  
Antonella Pinto ◽  
Rose-Marie Ölander ◽  
Dorothea Sesardic ◽  
Paul Stickings ◽  
...  
Keyword(s):  
Quality Assessment ◽  
External Quality Assessment ◽  
Accurate Determination ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reference Laboratory ◽  
Routine Method ◽  
External Quality ◽  
Passive Hemagglutination

ABSTRACT Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro “gold standard.” The performance of laboratories using NT was generally very good, while the laboratories’ performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

scholarly journals In Vitro and In Vivo Fitness of Respiratory Syncytial Virus Monoclonal Antibody Escape Mutants

2006 ◽  
Vol 80 (23) ◽  
pp. 11651-11657 ◽  
Author(s):  
Xiaodong Zhao ◽  
Enmei Liu ◽  
Fu-Ping Chen ◽  
Wayne M. Sullender
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibody ◽  
Respiratory Syncytial Virus ◽  
Viral Population ◽  
Nucleotide Substitutions ◽  
Cotton Rats ◽  
Syncytial Virus ◽  
In Vivo Growth

ABSTRACT Respiratory syncytial virus (RSV) is the only infectious disease for which a monoclonal antibody (MAb) is used in humans. Palivizumab (PZ) is a humanized murine MAb to the F protein of RSV. PZ-resistant viruses appear after in vitro and in vivo growth of RSV in the presence of PZ. Fitness for replication could be a determinant of the likelihood of dissemination of resistant viruses. We assessed the fitness of two PZ-resistant viruses (F212 and MP4). F212 grew less well in cell culture than the parent A2 virus and was predicted to be less fit than A2. Equal amounts of F212 and A2 were mixed and passaged in cell culture. F212 disappeared from the viral population, indicating it was less fit than the A2 virus. The MP4 virus grew as well as A2 in culture and in cotton rats. A2/MP4 virus input ratios of 1:1, 10:1, 100:1, and 1,000:1 were compared in competitive replication. For all input ratios except 1,000:1, the MP4 virus became dominant, supplanting the A2 virus. The MP4 virus also dominated the A2 virus during growth in cotton rats. Thus, the mutant MP4 virus was more fit than A2 virus in both in vitro and in vivo competitive replication. Whether this fitness difference was due to the identified nucleotide substitutions in the F gene or to mutations elsewhere in the genome is unknown. Understanding the mechanisms by which mutant virus fitness increased or decreased could prove useful for consideration in attenuated vaccine design efforts.

scholarly journals Genome-Wide Essentiality Analysis of Mycobacterium abscessus by Saturated Transposon Mutagenesis and Deep Sequencing

mBio ◽  
2021 ◽  
Author(s):  
Dalin Rifat ◽  
Liang Chen ◽  
Barry N. Kreiswirth ◽  
Eric L. Nuermberger
Keyword(s):  
Deep Sequencing ◽  
Mycobacterium Abscessus ◽  
Transposon Mutagenesis ◽  
Comprehensive Analysis ◽  
Data Sets ◽  
Similar Data ◽  
Intrinsic Resistance ◽  
Effective Strategies ◽  
Genome Wide

Limited knowledge regarding Mycobacterium abscessus pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify M. abscessus genetic elements essential for in vitro growth and compare them to similar data sets for M. tuberculosis and M. avium subsp. hominissuis .

scholarly journals 175 The Suitability of Autoclave and Pressure Cooker Washing Methods in Comparison to ANKOM200 to Determine Neutral Detergent Fiber Using Different Feedstuffs and Washing Solutions

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 79-79
Author(s):  
Jordan Adams ◽  
Aaron B Norris ◽  
Madeline E Rivera ◽  
Luiz Fernando Dias Batista ◽  
Luis O Tedeschi
Keyword(s):  
Accurate Determination ◽  
Gas Production ◽  
Random Coefficients ◽  
Neutral Detergent Fiber ◽  
Detergent Solution ◽  
Standard Methodology ◽  
Randomized Design ◽  
Factor Combination ◽  
High Forage

Abstract The use of the in vitro gas production (IVGP) technique requires accurate determination of neutral detergent fiber (NDF) residue. However, the NDF determination using standard procedures are not always feasible for IVGP; thus requiring micro-NDF methods, which need autoclave (AC) and pressure cooker (PC) to boil the solution. A complete randomized design using a 3×3 factorial arrangement was implemented to investigate the effect of washing methods (WM: AC, PC, or ANKOM200) and solution ratios (WS: 100 mL neutral detergent solution (ND), 150 mL ND, or 100 mL H2O/g sample) to determine NDF residues, assuming ANKOM200 and 100 mL ND/g as the standard methodology. Each factor combination was performed in triplicate with a replicate being comprised of 12 bottles or bags (two blanks and five feedstuffs in duplicate). Feedstuffs were: alfalfa hay (AH), bermudagrass hay (BH), two high-forage rations (G1 and G2), and a high-concentrate ration (FR). Following each run, bottles were filtered to obtain the NDF. Data were analyzed by diet using a random coefficients model. An interaction of WM ′ WS was present for AH and G1 (P < 0.01), G2 and FR had tendencies (P = 0.08 and 0.06, respectively), whereas BH demonstrated no interaction (P = 0.37). The PC with 100 mL or 150 mL did not differ from the standard methodology for AH, G1, G2, and FR. The BH demonstrated differences between WM and WS (P < 0.01). The PC had lower NDF residue compared to the AC and ANKOM200, whereas H2O had substantially greater NDF residue relative to both ND ratios. We concluded that H2O is not a suitable substitute for ND solution regardless of the feedstuff. Both micro-NDF washing methods may be satisfactory depending on the type of feedstuff used but further investigation is required.

scholarly journals Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments

1999 ◽  
Vol 73 (3) ◽  
pp. 2222-2231 ◽  
Author(s):  
Paul Digard ◽  
Debra Elton ◽  
Konrad Bishop ◽  
Elizabeth Medcalf ◽  
Alan Weeds ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Nuclear Localization ◽  
Virus Genome ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Nuclear Localization Signals ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
High Level

ABSTRACT The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.

scholarly journals Transcription of hepatitis B virus by RNA polymerase II.

1983 ◽  
Vol 3 (10) ◽  
pp. 1766-1773 ◽  
Author(s):  
L B Rall ◽  
D N Standring ◽  
O Laub ◽  
W J Rutter
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Simian Virus 40 ◽  
Virus Genome ◽  
Simian Virus ◽  
Free System ◽  
B Virus

We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.

scholarly journals Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

2009 ◽  
Vol 83 (10) ◽  
pp. 5137-5147 ◽  
Author(s):  
Hiromichi Hara ◽  
Hideki Aizaki ◽  
Mami Matsuda ◽  
Fumiko Shinkai-Ouchi ◽  
Yasushi Inoue ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Genome ◽  
Proteome Analysis ◽  
Virus Genome ◽  
Genome Replication ◽  
Creatine Kinase B ◽  
Efficient Replication

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

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