Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

ObjectiveIn 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization.MethodsRNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA.ResultsmNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject.ConclusionmNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.

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Pediatric Brainstem Encephalitis Outbreak Investigation with Metagenomic Next-Generation Sequencing

10.1101/414979 ◽

2018 ◽

Author(s):

Kristoffer E. Leon ◽

Didac Casas-Alba ◽

Akshaya Ramesh ◽

Lillian M. Khan ◽

Cristian Launes ◽

...

Keyword(s):

Next Generation Sequencing ◽

Phylogenetic Analyses ◽

P Value ◽

Brainstem Encephalitis ◽

Next Generation ◽

Single Nucleotide Variants ◽

Qrt Pcr ◽

Mcnemar’S Test ◽

Generation Sequencing ◽

Mcnemar's Test

AbstractIn 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in peripheral body sites, but EV was rarely identified in cerebrospinal fluid (CSF). RNA was extracted from CSF (n=20), plasma (n=9), stool (n=15) and nasopharyngeal samples (n=16) from 10 children with brainstem encephalitis or encephalomyelitis and 10 contemporaneous pediatric controls with presumed viral meningitis or encephalitis. Unbiased complementary DNA libraries were sequenced, and microbial pathogens were identified using a custom bioinformatics pipeline. Full-length virus genomes were assembled for phylogenetic analyses. Metagenomic next-generation sequencing (mNGS) was concordant with qRT-PCR for all samples positive by PCR (n=25). In virus-negative samples (n=35), mNGS detected virus in 28.6% (n=10), including 5 CSF samples. mNGS co-detected EV-A71 and another EV in 5 patients. Overall, mNGS increased the proportion of EV-positive samples from 42% (25/60) to 57% (34/60) (McNemar’s test; p-value = 0.0077). For CSF, mNGS doubled the number of pathogen-positive samples (McNemar’s test; p-value = 0.074). Using phylogenetic analysis, the outbreak EV-A71 clustered with a neuroinvasive German EV-A71 isolate. Brainstem encephalitis specific, non-synonymous EV-A71 single nucleotide variants were not identified. mNGS demonstrated 100% concordance with clinical qRT-PCR of EV-related brainstem encephalitis and significantly increased the detection of enteroviruses. Our findings increase the probability that neurologic complications observed were virus-induced rather than para-infectious. A comprehensive genomic analysis confirmed that the EV-A71 outbreak strain was closely related to a neuroinvasive German EV-A71 isolate. There were no clear-cut viral genomic differences that discriminated between patients with differing neurologic phenotypes.

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Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.662352 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiejun Peng ◽

Shan Bu ◽

Yueyan Yin ◽

Mengying Hua ◽

Kuangjie Zhao ◽

...

Keyword(s):

Yunnan Province ◽

Genetic Characterization ◽

Biological Properties ◽

Capsicum Frutescens ◽

Chain Reaction ◽

Positive Sense ◽

Polymerase Chain ◽

Pepper Vein Yellows Virus ◽

Generation Sequencing

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with some viruses of the genus Polerovirus. Pod pepper vein yellows virus (PoPeVYV) was recently reported as a new recombinant polerovirus causing interveinal yellowing, stunting, and leaf rolling in Capsicum frutescens plants at Wenshan city, Yunnan province, China. The complete genome sequence of its associated RNA has now been determined by next-generation sequencing and reverse transcription (RT) polymerase chain reaction (PCR). PoPeVYV-associated RNA (PoPeVYVaRNA) (GenBank Accession No. MW323470) has 2970 nucleotides and is closely related to other group II tlaRNAs, particularly tobacco bushy top disease-associated RNA (TBTDaRNA, GenBank Accession No. EF529625). In infection experiments on Nicotiana benthamiana and C. frutescens plants, synergism between PoPeVYVaRNA and PoPeVYV was demonstrated, leading to severe interveinal yellowing of leaves and stunting of plants. The results provide further information on the genetic and biological properties of the various agents associated with pepper vein yellows disease (PeVYD).

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Maternal Circulating Exosomal miRNAs as Non-invasive Biomarkers for the Prediction of Fetal Ventricular Septal Defect

Frontiers in Genetics ◽

10.3389/fgene.2021.717208 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuxia Jin ◽

Ling Ai ◽

Xiaojun Chai ◽

Ping Tang ◽

Weihua Zhang ◽

...

Keyword(s):

Predictive Biomarkers ◽

Roc Curves ◽

Control Group ◽

Differential Abundance ◽

Non Invasive ◽

Qrt Pcr ◽

Septal Defects ◽

Exosomal Mirnas ◽

Polymerase Chain ◽

Generation Sequencing

Objective: This study aimed to identify maternal circulating exosomal miRNAs as potential non-invasive biomarkers for the early detection of fetal ventricular septal defects (VSDs).Methods: In total, 182 pregnant women, comprising 91 VSD cases and 91 matched controls, were included in this study. Exosomes were isolated; dysregulated exosomal miRNAs were profiled using next-generation sequencing. Differential abundance of miRNAs was verified using quantitative real-time polymerase chain reaction (qRT-PCR). Diagnostic accuracy was evaluated by constructing receiver operating characteristic (ROC) curves.Results: In total, 77 serum exosomal miRNAs were found to be differentially expressed in the VSD group compared to their expression in the control group. Among these, five downregulated exosomal miRNAs were validated using qRT-PCR. hsa-miR-146a-5p was identified to be capable of distinguishing VSD cases from controls (area under the ROC curve [AUC]: 0.997; p < 1.00E-05).Conclusion: Circulating exosomal miRNAs, particularly hsa-miR-146a-5p, may be predictive biomarkers for the non-invasive prenatal diagnosis of fetal VSDs.

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Biochemical characterization of bovine αs1-casein F and genotyping with sequence-specific primers

Journal of Dairy Research ◽

10.1017/s0022029997002690 ◽

1998 ◽

Vol 65 (2) ◽

pp. 223-231 ◽

Cited By ~ 3

Author(s):

EVA-MARIA PRINZENBERG ◽

PATRICIA ANGLADE ◽

BRUNO RIBADEAU-DUMAS ◽

GEORG ERHARDT

Keyword(s):

Gene Sequence ◽

Genetic Resource ◽

Biochemical Characterization ◽

Reversed Phase ◽

Single Amino Acid ◽

Specific Primers ◽

New Variant ◽

Allele Specific ◽

Polymerase Chain

Bovine αs1-casein F (αs1-CN F) was found in a genetic resource of Deutsches Schwarzbuntes Niederungsrind cows at a frequency of 0·009. Biochemical characterization of this new variant was obtained by automated sequencing of reversed-phase HPLC-separated tryptic peptides of αs1-CN F and αs1-CN B. αs1-CN F was found to be a subtype of αs1-CN B with a single amino acid substitution (SerP/Leu) in position 66. DNA sequencing revealed a C/T transition in position 8418 of the gene. Sequence-specific primers were designed to perform an allele-specific polymerase chain reaction for detection of αs1CnF. Typing of artificial insemination sperm samples included in the genetic resource sperm pool identified one sire heterozygous for αs1CnF.

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Next generation sequencing and functional characterization of the androgen receptor (AR) gene in patients with androgen insensitivity syndrome (AIS) and controls

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-0035-1547779 ◽

2015 ◽

Vol 122 (03) ◽

Author(s):

N Hornig ◽

HU Schweikert ◽

M Ukat ◽

AE Kulle ◽

M Welzel ◽

...

Keyword(s):

Androgen Receptor ◽

Next Generation Sequencing ◽

Functional Characterization ◽

Androgen Insensitivity Syndrome ◽

Next Generation ◽

Androgen Insensitivity ◽

Generation Sequencing

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Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649885 ◽

1995 ◽

Vol 74 (04) ◽

pp. 1079-1087 ◽

Cited By ~ 8

Author(s):

Klaus-P Radtke ◽

José A Fernández ◽

Bruno O Villoutreix ◽

Judith S Greengard ◽

John H Griffin

Keyword(s):

Rhesus Monkey ◽

Protein C ◽

Activated Protein C ◽

Pcr Amplification ◽

Amino Acid Sequences ◽

Heparin Binding ◽

Reactive Center ◽

Protein C Inhibitor ◽

Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

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Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648381 ◽

1992 ◽

Vol 67 (01) ◽

pp. 063-065 ◽

Cited By ~ 6

Author(s):

Sherryl A M Taylor ◽

Jacalyn Duffin ◽

Cherie Cameron ◽

Jerome Teitel ◽

Bernadette Garvey ◽

...

Keyword(s):

Nucleotide Substitution ◽

Novel Mutation ◽

Factor Ix ◽

Causative Mutation ◽

Coding Region ◽

Body Of Knowledge ◽

Polymerase Chain ◽

Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

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