Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

ABSTRACTNeuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.

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Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Applied and Environmental Microbiology ◽

10.1128/aem.03934-13 ◽

2014 ◽

Vol 80 (7) ◽

pp. 2125-2132 ◽

Cited By ~ 23

Author(s):

Narjol Gonzalez-Escalona ◽

Ruth Timme ◽

Brian H. Raphael ◽

Donald Zink ◽

Shashi K. Sharma

Keyword(s):

Single Nucleotide Polymorphism ◽

Clostridium Botulinum ◽

Toxin Gene ◽

Polymorphism Analysis ◽

Snp Analysis ◽

Whole Genome ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Content Type ◽

Group I

ABSTRACTClostridium botulinumis a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA+OrfX−) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA−OrfX+) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producingC. botulinumstrains: two strains with the HA+OrfX−cluster (69A and 32A) and one strain with the HA−OrfX+cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly availableC. botulinumgroup I strains revealed five distinct lineages. Strains 69A and 32A clustered with theC. botulinumtype A1 Hall group, and strain CDC297 clustered with theC. botulinumtype Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination ofC. botulinumgroup I strains and demonstrates the utility of this analysis in quickly differentiatingC. botulinumstrains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

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Genotyping Schemes for Polyomavirus BK, Using Gene-Specific Phylogenetic Trees and Single Nucleotide Polymorphism Analysis

Journal of Virology ◽

10.1128/jvi.02180-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2285-2297 ◽

Cited By ~ 44

Author(s):

Chunqing Luo ◽

Marta Bueno ◽

Jeffrey Kant ◽

Jeremy Martinson ◽

Parmjeet Randhawa

Keyword(s):

Single Nucleotide Polymorphism ◽

Phylogenetic Trees ◽

Genetic Recombination ◽

T Antigen ◽

Polymorphism Analysis ◽

Snp Analysis ◽

Whole Genome ◽

Vp1 Gene ◽

Nucleotide Polymorphism ◽

Single Nucleotide

ABSTRACT BK virus (BKV) genotyping has been historically based on nucleotides 1744 to 1812 in the VP1 gene. We reevaluated this practice by making BKV whole-genome and gene-specific phylogenetic trees as well as performing single nucleotide polymorphism (SNP) analysis of 162 sequences available in the public domain. It was found that currently known BKV subtypes and subgroups can no longer be reliably determined by sequencing certain partial gene sequences. Phylogenetic trees based on large T-antigen (LTA) allow separation of subtype I into subgroups Ia, Ib1, Ib2, and Ic, with bootstrap values of 100%, which are better than bootstraps obtained using VP1 sequences (bootstrap values of 71 to 97%). Subtype IV can be subdivided into subgroups, but LTA bootstrap values (33 to 80%) are lower than those obtained by whole-genome analysis (68 to 87%). Subtypes V and VI provisionally identified earlier on the basis of more limited sequence data are better classified as subgroups Ib2 and Ib1, respectively. LTA positions 3634, 3772, 3934, and 4339 can serve as a minimal SNP set to distinguish between the four major BKV subtypes. No subtype II-, IVa-, or IVb-defining SNPs are available in the VP1 gene. However, the overall congruence of viral strain classification based on either VP1 or LTA phylogenetic analysis indicates that these two areas of the viral genome are genetically linked. Interstrain genetic recombination between distant loci in the VP1 and LTA areas is not a common event.

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Single Nucleotide Polymorphism Analysis in HIV and Kaposi's Sarcoma Disease by Microarray Technique

Current HIV Research ◽

10.2174/1570162x18666200130100654 ◽

2020 ◽

Vol 18 (3) ◽

pp. 154-164 ◽

Cited By ~ 1

Author(s):

Ismail Koyuncu ◽

Ataman Gönel ◽

Emrah Ozcan ◽

Ebru Temiz ◽

Şahin Toprak ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Response To Treatment ◽

Polymorphism Analysis ◽

Snp Analysis ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Blood Samples ◽

Disease Response

Background: Emergence of Kaposi's Sarcoma in the cases other than HIV, following the use of immunosuppressant drugs, demonstrates that it is related to weak immunity. The fact that this malignancy does not occur in every HIV-positive patient suggests that genetic predisposition may also be effective. Replacement of one of the base pairs of adenine, guanine, cytosine, and thymine that constitute the DNA sequence in the human genome with another base pair can affect susceptibility to disease, response to treatment, and immunity. Objective: The purpose of this study is to analyze the Single Nucleotide Polymorphism that could predispose to Kaposi's sarcoma of an HIV-infected patient and to identify which nucleotides such SNPs correspond to, using the microarray technology. Material and Method: The blood samples of individuals, one of whom was diagnosed with Kaposi's Sarcoma HIV (+) visiting the outpatient clinic of infectious diseases polyclinic of Harran University Research and Practice Hospital and of a healthy individual with no Kaposi's Sarcoma, were used in the study. Following the DNA isolation of the blood samples taken from the respective individuals, a SNP analysis was conducted on the microarray device. 204,000 SNPs obtained were scanned later on in the databases in an attempt to identify the SNPs related to Kaposi's Sarcoma. Results: In the 204,000 SNP screenings, we scrutinized the SNPs that differ in the case of Kaposi's Sarcoma [KS (+) and HIV (+)] on the basis of Control [KS(-) and HIV(-)] and HIV+ [KS(-)], and two SNPs of the ENDRA gene, three SNPs of the ADRA1A gene, six SNPs of the STIM1 gene, four SNPs of the EFNB2 gene, and one SNP of the CD209 gene were found to be different. However, when it comes to all SNPs (all the 204.000 SNPs) screened in terms of allele, it was observed that the AA and BB alleles were lower in the patient with Kaposi's Sarcoma [KS (+) and HIV (+)] compared to other groups and AB alleles were found to be higher than others in the patient with Kaposi's sarcoma [KS] (+) and HIV (+)]. Conclusion: In the microarray study we have conducted, 204,000 SNPs were screened for Control (HIV-) HIV (+) and HIV (+) patient with Kaposi's Sarcoma. It was found that 32,362 of those SNPs had different alleles in the Kaposi's Sarcoma [KS + HIV (+)] patient, while they had the same ones in the control [KS (-) and HIV (-)] and HIV + [KS (-)] group. 16 of the 32,362 SNPs took place among the genes related to Kaposi's Sarcoma. In the cases of Kaposi's Sarcoma with suspected diagnosis, it can be used as a beneficial laboratory test.

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Loop-mediated Isothermal Amplification-Single Nucleotide Polymorphism Analysis for Detection and Differentiation of Wild-type and Vaccine Strains of Mink Enteritis Virus

Scientific Reports ◽

10.1038/s41598-018-26717-6 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 6

Author(s):

Peng Lin ◽

Honglin Wang ◽

Yuening Cheng ◽

Shanshan Song ◽

Yaru Sun ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Isothermal Amplification ◽

Polymorphism Analysis ◽

Single Nucleotide Polymorphism Analysis ◽

Wild Type ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Loop Mediated Isothermal Amplification ◽

Mink Enteritis Virus ◽

Enteritis Virus

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Core Genome Multi Locus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

10.1101/297010 ◽

2018 ◽

Author(s):

Anna Janowicz ◽

Fabrizio De Massis ◽

Massimo Ancora ◽

Cesare Cammà ◽

Claudio Patavino ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Core Genome ◽

Brucella Melitensis ◽

Reference Sequence ◽

Polymorphism Analysis ◽

Snp Analysis ◽

Phylogenetic Distance ◽

Sample Collection ◽

Nucleotide Polymorphism ◽

Single Nucleotide

AbstractThe use of whole genome sequencing (WGS) using next generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through Single Nucleotide Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing (cgMLST) has not been explored so far. The current study developed an allele-based method cgMLST and compared its performance to the genome-wide SNP approach and the traditional MLVA on a defined sample collection. The dataset comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of the complete genome). We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤4 loci in the cgMLST and ≤10 in WGS SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance resolution than MLVA, particularly for strains belonging to the same lineage thus allowing diverse and unrelated genotypes to be identified with greater confidence. The application of this cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

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