scholarly journals Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in the Listeria monocytogenes Virulence-Associated Gene inlA

2008 ◽  
Vol 74 (23) ◽  
pp. 7365-7375 ◽  
Author(s):  
A. Van Stelten ◽  
K. K. Nightingale
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Listeria Monocytogenes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Snp Genotyping ◽  
Regulatory Agencies ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Genotyping Assay ◽  
Virulence Attenuation

ABSTRACT The virulence factor internalin A (InlA) facilitates the uptake of Listeria monocytogenes by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in inlA and demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700 L. monocytogenes isolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing of inlA genes from isolates with attenuated invasion capacities revealed three novel inlA PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without inlA PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in inlA. The SNP genotyping assay detects all inlA PMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novel inlA PMSC mutations. PMSCs in inlA were significantly (ribotypes DUP-1039C and DUP-1045B; P < 0.001) or marginally (ribotype DUP-1062D; P = 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S. L. monocytogenes isolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiate L. monocytogenes isolates carrying virulence-attenuating PMSC mutations in inlA from strains representing the most significant health risk.

scholarly journals Revelation by Single-Nucleotide Polymorphism Genotyping That Mutations Leading to a Premature Stop Codon in inlA Are Common among Listeria monocytogenes Isolates from Ready-To-Eat Foods but Not Human Listeriosis Cases

2010 ◽  
Vol 76 (9) ◽  
pp. 2783-2790 ◽  
Author(s):  
A. Van Stelten ◽  
J. M. Simpson ◽  
T. J. Ward ◽  
K. K. Nightingale
Keyword(s):  
United States ◽  
Single Nucleotide Polymorphism ◽  
Listeria Monocytogenes ◽  
Stop Codon ◽  
Human Health Risk ◽  
Premature Stop Codon ◽  
The United States ◽  
Nucleotide Polymorphism ◽  
Pathogenic Potential ◽  
Single Nucleotide

ABSTRACT Listeria monocytogenes utilizes internalin A (InlA; encoded by inlA) to cross the intestinal barrier to establish a systemic infection. Multiple naturally occurring mutations leading to a premature stop codon (PMSC) in inlA have been reported worldwide, and these mutations are causally associated with attenuated virulence. Five inlA PMSC mutations recently discovered among isolates from France and the United States were included as additional markers in our previously described inlA single-nucleotide polymorphism (SNP) genotyping assay. This assay was used to screen >1,000 L. monocytogenes isolates from ready-to-eat (RTE) foods (n = 502) and human listeriosis cases (n = 507) for 18 inlA PMSC mutations. A significantly (P < 0.0001) greater proportion of RTE food isolates (45.0%) carried a PMSC mutation in inlA compared to human clinical isolates (5.1%). The proportion of L. monocytogenes with or without PMSC mutations in inlA was similar among isolates from different RTE food categories except for deli meats, which included a marginally higher proportion (P = 0.12) of isolates carrying a PMSC in inlA. We also analyzed the distribution of epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States. We observed a significant (P < 0.05) overrepresentation of EC strains in deli and seafood salads and a significant (P < 0.05) underrepresentation of EC strains in smoked seafood. These results provide important data to predict the human health risk of exposure to L. monocytogenes strains that differ in pathogenic potential through consumption of contaminated RTE foods.

scholarly journals Single Nucleotide Polymorphism Genotyping Analysis Shows That Vaccination Can Limit the Number and Diversity of Recombinant Progeny of Infectious Laryngotracheitis Viruses from the United States

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Carlos A. Loncoman ◽  
Carol A. Hartley ◽  
Mauricio J. C. Coppo ◽  
Glenn F. Browning ◽  
Gabriela Beltrán ◽  
...  
Keyword(s):  
United States ◽  
Single Nucleotide Polymorphism ◽  
Viral Replication ◽  
The United States ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Genotyping Assay ◽  
Infectious Laryngotracheitis

ABSTRACT Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) causes mild to severe respiratory disease in poultry worldwide. Recombination in this virus under natural (field) conditions was first described in 2012 and more recently has been studied under laboratory conditions. Previous studies have revealed that natural recombination is widespread in ILTV and have also demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In the United States, natural ILTV recombination has also been detected, but not as frequently as in Australia. To better understand recombination in ILTV strains originating from the United States, we developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two virulent U.S. field strains of ILTV (63140 and 1874c5) under experimental in vivo conditions. We also tested the capacity of the Innovax-ILT vaccine (a recombinant vaccine using herpesvirus of turkeys as a vector) and the Trachivax vaccine (a conventionally attenuated chicken embryo origin vaccine) to reduce recombination. The Trachivax vaccine prevented ILTV replication, and therefore recombination, in the trachea after challenge. The Innovax-ILT vaccine allowed the challenge viruses to replicate and to recombine, but at a significantly lower rate than in an unvaccinated group of birds. Our results demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination between these ILTV strains and also show that vaccination can limit the number and diversity of recombinant progeny viruses. IMPORTANCE Recombination allows alphaherpesviruses to evolve over time and become more virulent. Historically, characterization of viral vaccines in poultry have mainly focused on limiting clinical disease, rather than limiting virus replication, but such approaches can allow field viruses to persist and evolve in vaccinated populations. In this study, we vaccinated chickens with Gallid alphaherpesvirus 1 vaccines that are commercially available in the United States and then performed coinoculations with two field strains of virus to measure the ability of the vaccines to prevent field strains from replicating and recombining. We found that vaccination reduced viral replication, recombination, and diversity compared to those in unvaccinated chickens, although the extent to which this occurred differed between vaccines. We suggest that characterization of vaccines could include studies to examine the ability of vaccines to reduce viral recombination in order to limit the rise of new virulent field strains due to recombination, especially for those vaccines that are known not to prevent viral replication following challenge.

Analysis of Additional Virulence Genes and Virulence Gene Regions in Listeria monocytogenes Confirms the Epidemiologic Relevance of Multi-Virulence-Locus Sequence Typing

2008 ◽  
Vol 71 (12) ◽  
pp. 2559-2566 ◽  
Author(s):  
SARA LOMONACO ◽  
YI CHEN ◽  
STEPHEN J. KNABEL
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Listeria Monocytogenes ◽  
Virulence Genes ◽  
Virulence Gene ◽  
The Other ◽  
Evolutionary Divergence ◽  
Gene Sequences ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Molecular Subtyping

Previous molecular subtyping studies have defined four epidemic clones (ECs) of Listeria monocytogenes (ECI, ECII, ECIII, and ECIV). Partial sequences of eight virulence genes were previously shown to be identical within individual ECs of L. monocytogenes. The present study was conducted to determine if the sequences of other virulence genes and virulence gene regions are also conserved within these ECs. Six additional virulence genes—bsh, hly, inlJ, lplA1, pgdA, and srtA—and three additional virulence gene regions of actA, inlA, and inlB were selected based on their role in L. monocytogenes virulence, and intragenic regions of each gene were sequenced. Sequencing was performed on a diverse set of 44 to 48 L. monocytogenes strains. Results demonstrated that the sequenced regions of the nine virulence genes were identical within each of the ECs, and 257 new single nucleotide polymorphism (SNPs) were identified. ECIII (lineage II) was easily distinguishable from the other ECs, as 238 SNPs were observed in ECIII due to its significant evolutionary divergence from lineage I. With regard to the other ECs, there were 5 SNPs that represented an informative set, since these SNPs were able to differentiate specific ECs from all other unrelated strains used in this study. This study confirms our previous finding that virulence gene sequences are highly conserved within individual ECs and contain stable SNPs that can be used to very accurately differentiate ECs of L. monocytogenes from each other and from other diverse strains.

Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution

2009 ◽  
Vol 4 (6) ◽  
pp. 984-991 ◽  
Author(s):  
Xinrui Duan ◽  
Wei Yue ◽  
Libing Liu ◽  
Zhengping Li ◽  
Yuliang Li ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Conjugated Polymers ◽  
Homogeneous Solution ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

scholarly journals Single-Nucleotide-Polymorphism Genotyping ofCoxiella burnetiiduring a Q Fever Outbreak in The Netherlands

2011 ◽  
Vol 77 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
Cornelis J. J. Huijsmans ◽  
Jeroen J. A. Schellekens ◽  
Peter C. Wever ◽  
Rudolf Toman ◽  
Paul H. M. Savelkoul ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
The Netherlands ◽  
Q Fever ◽  
Snp Genotyping ◽  
Cow Milk ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Animal Reservoir ◽  
Single Nucleotide ◽  
Outbreak Area

ABSTRACTCoxiella burnetiiis the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of theC. burnetiistrain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination ofC. burnetiistrains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 differentC. burnetiigenotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 differentC. burnetiigenotypes are involved in the Dutch outbreak.

scholarly journals Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

2017 ◽  
Vol 85 (11) ◽  
Author(s):  
Mylène M. Maury ◽  
Viviane Chenal-Francisque ◽  
Hélène Bracq-Dieye ◽  
Lei Han ◽  
Alexandre Leclercq ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Host Cells ◽  
Single Amino Acid ◽  
Listeriolysin O ◽  
Loss Of Function ◽  
Phenotypic Marker ◽  
Content Type ◽  
Virulence Attenuation

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.

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