Full genome of influenza A (H7N9) virus derived by direct sequencing without culture.
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ABSTRACT:
SUBMITTER: Ren X
PROVIDER: S-EPMC3837655 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We analyzed the complete genome sequence of the A/Shanghai/01/2014 (H7N9) strain, which will provide a better understanding of the evolution of influenza A(H7N9) virus.
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Project description:The aim of the present study was to analyze the evolution and variation of a novel strain of the avian influenza virus. The virus-positive specimens [A/Changsha/2/2013 (H7N9)] from a patient infected with the novel avian influenza A (H7N9) virus was amplified by reverse transcription-PCR and the full genome was sequenced. The sequencing results were submitted to GenBank and then analyzed by phylogenetic tree analysis using BioEdit and Mega5 software. The phylogenetic tree of the hemagglutinin (HA) and neuraminidase genes revealed that A/Changsha/2/2013 (H7N9) and all the new H7N9 viruses in 2013 were in a large cluster, and their nucleotide evolutionary distances were closely associated. Phylogenetic tree analyses of the nucleoprotein and nonstructural genes demonstrated two main branches. One branch contained novel H7N9 viruses isolated from avian, human and environmental sources in different regions. The other branch contained three novel H7N9 virus strains isolated from environmental sources in Shanghai. All the phylogenetic trees of the matrix protein, polymerase acidic, polymerase basic protein 1 and polymerase basic protein 2 genes also showed two branches, with each branch including the novel H7N9 virus strains isolated from avian, human and environmental sources in different regions. Molecular characterization demonstrated that 52 novel H7N9 viruses sequenced to date contain the G228S and G186V mutations in the receptor binding site of the HA protein. The full-genome sequences of A/Changsha/2/2013 and analyses of its molecular characteristics suggest that the A/Changsha/2/2013 H7N9 virus strain has molecular characteristics that may facilitate adaptation of the virus to mammalian hosts and may even bind to human receptors.
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Project description:For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 × 109), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.
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Project description:Next-generation sequencing (NGS) enables the recovery of pathogen genomes from clinical samples without the need for culturing. Depletion of host/microbiota components (e.g., ribosomal RNA and poly-A RNA) and whole DNA/cDNA amplification are routine methods to improve recovery results. Using mixtures of human and influenza A virus (H1N1) RNA as a model, we found that background depletion and whole transcriptome amplification introduced biased distributions of read coverage over the H1N1 genome, thereby hampering genome assembly. Influenza serotyping was also affected by pretreatments. We propose that direct sequencing of noncultured samples without pretreatment is a favorable option for pathogen genome recovery applications.
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Project description:Influenza A virus strain:H7N9 Genome sequencing
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Project description:Without baseline human immunity to the emergent avian influenza A(H7N9) virus, neuraminidase inhibitors are vital for controlling viral replication in severe infections. An amino acid change in the viral neuraminidase associated with drug resistance, NA-R292K (N2 numbering), has been found in some H7N9 clinical isolates. Here we assess the impact of the NA-R292K substitution on antiviral sensitivity and viral replication, pathogenicity and transmissibility of H7N9 viruses. Our data indicate that an H7N9 isolate encoding the NA-R292K substitution is highly resistant to oseltamivir and peramivir and partially resistant to zanamivir. Furthermore, H7N9 reassortants with and without the resistance mutation demonstrate comparable viral replication in primary human respiratory cells, virulence in mice and transmissibility in guinea pigs. Thus, in stark contrast to oseltamivir-resistant seasonal influenza A(H3N2) viruses, H7N9 virus replication and pathogenicity in these models are not substantially altered by the acquisition of high-level oseltamivir resistance due to the NA-R292K mutation.
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Project description:mRNA-Seq analysis was used to profile the cellular transcriptome of A549 cells at multiple time points in response to infection with influenza H7N9.
2017-04-22 | GSE97949 | GEO
Project description:Avian influenza virus A (H7N9), after circulating in avian hosts for decades, was identified as a human pathogen in 2013. Herein, amino acid substitutions possibly essential for human adaptation were identified by comparing the 4706 aligned overlapping nonamer position sequences (1-9, 2-10, etc.) of the reported 2014 and 2017 avian and human H7N9 datasets. The initial set of virus sequences (as of year 2014) exhibited a total of 109 avian-to-human (A2H) signature amino acid substitutions. Each represented the most prevalent substitution at a given avian virus nonamer position that was selectively adapted as the corresponding index (most prevalent sequence) of the human viruses. The majority of these avian substitutions were long-standing in the evolution of H7N9, and only 17 were first detected in 2013 as possibly essential for the initial human adaptation. Strikingly, continued evolution of the avian H7N9 virus has resulted in avian and human protein sequences that are almost identical. This rapid and continued adaptation of the avian H7N9 virus to the human host, with near identity of the avian and human viruses, is associated with increased human infection and a predicted greater risk of human-to-human transmission.
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Project description:Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation "nanopore" technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was > 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.
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Project description:Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 102 to 103 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P?=?4.7?×?10-5). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [CT ] values, <30) and 6/13 samples with low viral titers (CT values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct >99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.
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