A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses

Deng, Yi‐Mo; Spirason, Natalie; Iannello, Pina; Jelley, Lauren; Lau, Hilda; Barr, Ian G.

doi:10.1016/j.jcv.2015.04.019

Cited by 45 publications

(38 citation statements)

References 25 publications

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“…RNA extraction from the isolates was carried out using QIAmp Viral RNA Mini Kit (Qiagen, Inc., USA) according to the manufacturer's instructions. RT-PCR amplification of the HA gene was performed using Superscript III One Step RT-PCR System (Invitrogen Corporation, USA) and a set of previously described M13 tagged primers [31]. A final reaction mix of 25μL containing 2 X Reaction Mix, 20pmoles/μL of both Forward and Reverse primers, Superscript RT/Platinum Taq enzyme mix, Nuclease-free water (Promega Corporation, USA) and 3μL RNA template was prepared.…”

Section: Rna Extraction Rt-pcr and Sanger Sequencingmentioning

confidence: 99%

“…The PCR amplicons (frag 1: S 976 bp; frag 2: S 890 bp) were resolved on a 1% Agarose gel (Sigma-Aldrich Co., USA) stained with ethidium bromide (0.5 mg/ml) (Sigma-Aldrich Co., USA) and visualized using the Ebox gel documentation system (Vilber Lourmat, France) according to the manufacturer's instructions. The PCR products were cleaned using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, USA) and sequenced directly on both strands using universal M13 forward and reverse primers, as previously described [29,31]. Cycle sequencing was carried out using the Big Dye Terminator Cycle sequencing kit v3.1 (Applied Biosystems, USA) and products were resolved on an automated 3500xL Genetic Analyzer (Applied Biosystems, USA) according to the manufacturer's instructions.…”

Section: Rna Extraction Rt-pcr and Sanger Sequencingmentioning

confidence: 99%

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Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene

et al. 2020

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Influenza A (H1N1) pdm09 virus emerged in North America in 2009 and has been established as a seasonal strain in humans. After an antigenic stasis of about six years, new antigenically distinct variants of the virus emerged globally in 2016 necessitating a change in the vaccine formulation for the first time in 2017. Herein, we analyzed thirty-eight HA sequences of influenza A (H1N1) pdm09 strains isolated in Kenya during 2015-2018 seasons, to evaluate their antigenic and molecular properties based on the HA1 sub-unit. Our analyses revealed that the A (H1N1) pdm09 strains that circulated in Kenya during this period belonged to genetic clade 6B, subclade 6B.1 and 6B.2. The Kenyan 2015 and 2016 isolates differed from the vaccine strain A/California/07/2009 at nine and fourteen antigenic sites in the HA1 respectively. Further, those isolated in 2017 and 2018 correspondingly varied from A/Michigan/45/2015 vaccine strain at three and fifteen antigenic sites. The predicted vaccine efficacy of A/California/07/2009 against Kenyan 2015/2016 was estimated to be 32.4% while A/Michigan/45/2015 showed estimated vaccine efficacies of 39.6% -41.8% and 32.4% -42.1% against Kenyan 2017 and 2018 strains, respectively. Hemagglutinationinhibition (HAI) assay using ferret post-infection reference antiserum showed that the titers for the Kenyan 2015/2016 isolates were 2-8-fold lower compared to the vaccine strain. Overall, our results suggest the A (H1N1) pdm09 viruses that circulated in Kenya during 2015/2016 influenza seasons were antigenic variants of the recommended vaccine strains, denoting sub-optimal vaccine efficacy. Additionally, data generated point to a swiftly evolving influenza A (H1N1) pdm09 virus in recent post pandemic era, underscoring the need for sustained surveillance coupled with molecular and antigenic analyses, to inform appropriate and timely influenza vaccine update.

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Section: Rna Extraction Rt-pcr and Sanger Sequencingmentioning

confidence: 99%

Section: Rna Extraction Rt-pcr and Sanger Sequencingmentioning

confidence: 99%

Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene

et al. 2020

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“…Influenza A/Brisbane/10/2007 (H3N2) and influenza A/aquatic bird/Korea/w351/2008 (H5N2) suspended in cell culture medium (MEM, MDCK cells) were obtained from Korea Research Institute of Bioscience and Biotechnology, Korea. Each virus titer of the stock solutions ranged between 5 × 10 6 and 5 × 10 7 pfu mL −1 and was determined by real‐time reverse transcription polymerase chain reaction, according to a previous report . The monoclonal anti‐influenza A H1N1 antibody (ab128412) and goat anti‐rabbit IgG H&L (HRP) (ab6721) were purchased from Abcam.…”

Section: Methodsmentioning

confidence: 99%

Surface‐Independent and Oriented Immobilization of Antibody via One‐Step Polydopamine/Protein G Coating: Application to Influenza Virus Immunoassay

Moon

Byun

Kim

et al. 2019

Macromolecular Bioscience

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For the construction of high‐performance biosensor, it is important to interface bioreceptors with the sensor surface densely and in the optimal orientation. Herein, a simple surface modification method that can optimally immobilize antibodies onto various kinds of surfaces is reported. For the surface modification, a mixture of polydopamine (PDA) and protein G was employed. PDA is a representative mussel‐inspired polymer, and protein G is an immunoglobulin‐binding protein that enables an antibody to have an optimal orientation. The surface characteristics of PDA/Protein G mixture‐coated substrates are analyzed and the PDA/protein G ratio is optimized to maximize the antibody binding efficiency. Moreover, the antibody‐immobilized substrates are applied to the detection of influenza viruses with the naked eye, providing a detection limit of 2.9 × 103 pfu mL‐1. Importantly, the several substrates (glass, SiO2, Si, Al2O3, polyethylene terephthalate, polyethylene, polypropylene, and paper) can be modified by simple incubation with the mixture of PDA/protein G, and then the anti‐influenza A H1N1 antibodies can be immobilized on the substrates successfully. Regardless of the substrate, the influenza viruses are detectable after the sandwich immunoreaction and silver enhancement procedure. It is anticipated that the developed PDA/protein G coating method will extend the range of applicable materials for biosensing.

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“…Illumina MiSeq Next Generation Sequencer was employed to sequence the full genomes of influenza A, while FluSeq v1.0 was used for genome assembly (Lee et al, 2016). Sanger sequencing was employed to validate the HA1 segments of influenza A, and to sequence the genomes of influenza B. A/H3N2 primers (Lee et al, 2013), A/H1N1 primers (Deng et al, 2015) and 18 sets of B primers (Tewawong et al, 2015a) were used for sequencing. An additional set of primers (Chi et al, 2005) was also used for sequencing the HA1 genes of B viruses.…”

Section: Virus Propagation Rna Extraction and Genome Sequencingmentioning

confidence: 99%

Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore

Ivan

Zhou

Lau

et al. 2020

International Journal of Infectious Diseases

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A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses

Abstract: This streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols.

Cited by 45 publications

References 25 publications

Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene

Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene

Surface‐Independent and Oriented Immobilization of Antibody via One‐Step Polydopamine/Protein G Coating: Application to Influenza Virus Immunoassay

Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore

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