Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay

Abstract Background Artemisinin-resistant malaria (ARM) remains a significant threat to malaria elimination. In the Greater Mekong subregion, the prevalence of ARM in certain regions has reached greater than 90%. Artemisinin-resistant malaria is clinically identified by delayed parasite clearance and has been associated with mutations in the propeller domain of the kelch 13 gene. C580Y is the most prevalent mutation. The detection of ARM currently relies on labor-intensive and time-consuming methods such as clinical phenotyping or in vitro susceptibility testing. Methods We developed a novel single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) test method for the detection of the C580Y mutation using a novel primer design strategy. Results The SNP-LAMP was 90.0% sensitive (95% confidence interval [CI], 66.9–98.3) and 91.9% specific (95% CI, 82.6–96.7) without knowledge of the parasite load and was 100% sensitive (95% CI, 79.9–100) and 97.3% specific (95% CI, 89.7–99.5) when the parasitemia was within the assay dynamic range. Tests with potential application near-to-patient such as SNP-LAMP may be deployed in low- and middle-income and developed countries. Conclusions Single-nucleotide polymorphism LAMP can serve as a surveillance tool and guide treatment algorithms for ARM in a clinically relevant time frame, prevent unnecessary use of additional drugs that may drive additional resistance, and avoid longer treatment regimens that cause toxicity for the patient.

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Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

Scientific Reports ◽

10.1038/s41598-019-53124-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Michelle A. Land ◽

Holly L. Chapman ◽

Brionna D. Davis-Reyes ◽

Daniel E. Felsing ◽

John A. Allen ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Subcellular Localization ◽

Cell Lines ◽

Intracellular Signaling ◽

Calcium Release ◽

Wild Type ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

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Single-Nucleotide-Polymorphism-Specific PCR for Quantification and Discrimination of Chlamydia pneumoniae Genotypes by Use of a “Locked” Nucleic Acid

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3785-3787.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3785-3787 ◽

Cited By ~ 11

Author(s):

Jan Rupp ◽

Werner Solbach ◽

Jens Gieffers

Keyword(s):

Single Nucleotide Polymorphism ◽

Chlamydia Pneumoniae ◽

Locked Nucleic Acid ◽

Nucleotide Polymorphisms ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Specific Pcr ◽

Intraspecies Diversity

ABSTRACT Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

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A Novel Assay for Viral MicroRNA Function Identifies a Single Nucleotide Polymorphism That Affects Drosha Processing

Journal of Virology ◽

10.1128/jvi.02734-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5321-5326 ◽

Cited By ~ 97

Author(s):

Eva Gottwein ◽

Xuezhong Cai ◽

Bryan R. Cullen

Keyword(s):

Single Nucleotide Polymorphism ◽

Target Genes ◽

Human Herpesvirus ◽

Northern Analysis ◽

Luciferase Expression ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Stem Loop

ABSTRACT MicroRNAs (miRNAs) are a class of ∼22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3′ untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.

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