scholarly journals Human Cytomegalovirus Genome Diversity in Longitudinally Collected Breast Milk Samples

2021 ◽  
Vol 11 ◽  
Author(s):  
Jasper Götting ◽  
Katrin Lazar ◽  
Nicolás M. Suárez ◽  
Lars Steinbrück ◽  
Tabea Rabe ◽  
...  
Keyword(s):  
Breast Milk ◽  
Human Cytomegalovirus ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Severe Disease ◽  
Viral Population ◽  
Preterm Neonates ◽  
Genotype Distribution ◽  
Milk Samples ◽  
Wide Range

Reactivation and shedding of human cytomegalovirus (HCMV) in breast milk during lactation is highly frequent in HCMV-seropositive mothers. This represents a key transmission route for postnatal HCMV infection and can lead to severe disease in preterm neonates. Little is known about HCMV strain composition or longitudinal intrahost viral population dynamics in breast milk from immunocompetent women. We performed HCMV-specific target enrichment and high-throughput sequencing of 38 breast milk samples obtained in Germany between days 10 and 60 postpartum from 15 mothers with HCMV DNA lactia, and assembled HCMV consensus sequences de novo. The genotype distribution and number of HCMV strains present in each sample were determined by quantifying genotype-specific sequence motifs in 12 hypervariable viral genes, revealing a wide range of genotypes (82/109) for these genes in the cohort and a unique, longitudinally stable strain composition in each mother. Reactivation of up to three distinct HCMV strains was detected in 8/15 of mothers, indicating that a representative subset of the woman’s HCMV reservoir might be locally reactivated early during lactation. As described previously, nucleotide diversity of samples with multiple strains was much higher than that of samples with single strains. Breast milk as a main source of postnatal mother-to-infant transmission may serve as a repository for viral diversity and thus play an essential role in the natural epidemiology of HCMV.

Distribution of Human Cytomegalovirus Glycoprotein H Genotypes in Different Samples from Chinese Children

2021 ◽  
Author(s):  
Wei Li ◽  
Lu-Yan Chen ◽  
Ran Tao ◽  
Shi-Qiang Shang
Keyword(s):  
Breast Milk ◽  
Human Cytomegalovirus ◽  
Throat Swab ◽  
Quantitative Polymerase Chain Reaction ◽  
Age Groups ◽  
Urine Samples ◽  
Serum Samples ◽  
Predominant Genotype ◽  
Milk Samples ◽  
Glycoprotein H

Abstract Objective This study aimed to investigate characteristics of human cytomegalovirus (HCMV) glycoprotein H (gH) genotypes in urine, throat swab, and serum from children and breast milk from children's mothers. Methods Fresh urine samples, throat swabs, or serum samples from children and breast milk samples from children's mothers were collected for HCMV DNA detection. The positive samples of HCMV DNA were further detected by fluorescent quantitative polymerase chain reaction (PCR) with gH typing. Results Of 1,703 HCMV DNA-positive samples, the highest proportion (83.3%, 85/102) of children aged between 21 days and 3 months was detected positive in breast milk samples (p = 0.002), and the highest proportion (70.5%, 110/156) of children aged above 3 months was detected positive in throat swab samples (p = 0.002). HCMV in throat swab specimens is mainly high copy (p < 0.0001), and low-copy HCMV is prevalent in breast specimens (p < 0.0001). Among them, 1,059 samples were identified as gH1 genotype, 530 samples were gH2, and 114 samples were coinfection (gH1/2). There had the highest gH2 rates (32.3%) and lowest gH1 (61.0%) rates in urine samples (p = 0.041), whereas the highest gH1 rates (71.6%) and lowest gH2 rates (19.6%) were found in breast milk samples (p = 0.032). Concerning age groups, patients aged between 21 days and 3 months had the highest gH1 proportion (p = 0.017), while patients aged above 3 months had the highest gH1 and gH2 HCMV coinfection proportion (p = 0.002). Among 43 pairs of maternal and child samples corresponding to positive samples, gH genotype of 35 pairs of samples was consistent with a rate of 81.4%. Conclusion gH1 is the predominant genotype of HCMV in each kind of sample in China. However, the distribution of the HCMV gH genotype is different among different samples.

scholarly journals Fast sequence-based microsatellite genotyping development workflow

2019 ◽  
Author(s):  
Olivier Lepais ◽  
Emilie Chancerel ◽  
Christophe Boury ◽  
Franck Salin ◽  
Aurélie Manicki ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Ad Hoc ◽  
De Novo ◽  
Microsatellite Genotyping ◽  
Sequencing Technologies ◽  
Wide Range ◽  
Genetic Diversity And Structure ◽  
Genomic Resource ◽  
Analytical Frameworks ◽  
Different Levels

AbstractApplication of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20 to 40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence, quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

scholarly journals Human Breast Milk-acquired Cytomegalovirus Infection: Certainties, Doubts and Perspectives

2019 ◽  
Vol 15 (1) ◽  
pp. 30-41 ◽  
Author(s):  
Flaminia Bardanzellu ◽  
Vassilios Fanos ◽  
Alessandra Reali
Keyword(s):  
Breast Milk ◽  
Severe Disease ◽  
Neurological Impairment ◽  
Current Evidence ◽  
Preterm Neonates ◽  
Human Breast Milk ◽  
Increased Risk ◽  
Relevant Role ◽  
Beneficial Impact ◽  
Ultraviolet C

Breast Milk (BM) is the best source of nutrition for newborns, especially if premature. In fact, its beneficial impact on short- and the long-term neonatal outcome has was deeply described. Unfortunately, BM could not be always so safe, especially due to the possible presence of maternal viruses that can be shed and transferred to the breastfed neonate. Among these, Cytomegalovirus (CMV) can potentially lead to a serious and acute illness, mostly in case of low gestational age. Some studies also report the association of CMV-acquired infection to an increased risk of structural and functional brain modifications and neurological impairment. Due to these reasons, a strategy to remove CMV from BM with a minimal or absent impact on its beneficial components would be desirable. Up to now, pasteurization, freezing, ultraviolet- C or microwave irradiation are the available techniques; they show different levels of efficacy and variable effects on BM composition, even if many studies are still needed to fully clarify these implications. In this review, we provide an update of the current evidence about these topics. We focus on the factors promoting CMV shedding through BM; moreover, the possible occurrence of a severe disease in preterm neonates is also described. Finally, we investigate the potential effects showed on BM properties by the strategies that prevent or reduce viral transmission, therefore influencing newborns’ health, and the new techniques which could show a relevant role in the next future, such as metabolomics.

scholarly journals Multiple-Strain Infections of Human Cytomegalovirus With High Genomic Diversity Are Common in Breast Milk From Human Immunodeficiency Virus–Infected Women in Zambia

2019 ◽  
Vol 220 (5) ◽  
pp. 792-801 ◽  
Author(s):  
Nicolás M Suárez ◽  
Kunda G Musonda ◽  
Eric Escriva ◽  
Margaret Njenga ◽  
Anthony Agbueze ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Breast Milk ◽  
Human Cytomegalovirus ◽  
Developed Countries ◽  
Genomic Diversity ◽  
Viral Loads ◽  
Strain Diversity ◽  
Milk Samples ◽  
Immunodeficiency Virus ◽  
Hiv Negative

Abstract Background In developed countries, human cytomegalovirus (HCMV) is a major pathogen in congenitally infected and immunocompromised individuals, where multiple-strain infection appears linked to disease severity. The situation is less documented in developing countries. In Zambia, breast milk is a key route for transmitting HCMV and carries higher viral loads in human immunodeficiency virus (HIV)–infected women. We investigated HCMV strain diversity. Methods High-throughput sequence datasets were generated from 28 HCMV-positive breast milk samples donated by 22 mothers (15 HIV-infected and 7 HIV-negative) at 4–16 weeks postpartum, then analyzed by genome assembly and novel motif-based genotyping in 12 hypervariable HCMV genes. Results Among the 20 samples from 14 donors (13 HIV-infected and one HIV-negative) who yielded data meeting quality thresholds, 89 of the possible 109 genotypes were detected, and multiple-strain infections involving up to 5 strains per person were apparent in 9 HIV-infected women. Strain diversity was extensive among individuals but conserved compartmentally and longitudinally within them. Genotypic linkage was maintained within hypervariable UL73/UL74 and RL12/RL13/UL1 loci for virus entry and immunomodulation, but not between genes more distant from each other. Conclusions Breast milk from HIV-infected women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.

scholarly journals Multiple-Strain Infections of Human Cytomegalovirus with High Genomic Diversity are Common In Breast Milk from HIV-Positive Women in Zambia

2018 ◽  
Author(s):  
Nicolás M. Suárez ◽  
Kunda G. Musonda ◽  
Eric Escriva ◽  
Margaret Njenga ◽  
Anthony Agbueze ◽  
...  
Keyword(s):  
Breast Milk ◽  
Human Cytomegalovirus ◽  
Developed Countries ◽  
Genomic Diversity ◽  
Hiv Positive ◽  
Viral Loads ◽  
Strain Diversity ◽  
Milk Samples ◽  
Hiv Positive Women ◽  
Hiv Negative

ABSTRACTBackgroundIn developed countries, human cytomegalovirus (HCMV) is a major pathogen in congenitally infected and immunocompromised individuals, in whom multiple-strain infection is linked to disease severity. The situation is less documente in developing countries. In Zambia, breast milk is a key route for transmitting HCMV and carries higher viral loads in HIV-positive women. We investigated HCMV strain diversity.MethodsHigh-throughput sequence datasets were generated from 28 HCMV-positive breast milk samples donated by 22 mothers (15 HIV-positive and seven HIV-negative) at 4 or 16 weeks (or both) postpartum and analysed by genotyping 12 hypervariable HCMV genes.ResultsAmong the 20 samples from 14 donors (13 HIV-positive and one HIV-negative) that yielded data meeting quality thresholds, 89 of the possible 109 genotypes were detected, and multiple-strain infections involving up to five strains per person were apparent in nine HIV-positive women. Strain diversity was extensive among individuals but conserved compartmentally and longitudinally within them. Genotypic linkage was maintained within the hypervariable UL73/UL74 and RL12/RL13/UL1 loci for virus-entry and immunomodulation, but not between genes more distant from each other.ConclusionsBreast milk from HIV-positive women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.

scholarly journals Unravelling the virome in birch: RNA-Seq reveals a complex of known and novel viruses

2019 ◽  
Author(s):  
Artemis Rumbou ◽  
Thierry Candresse ◽  
Armelle Marais ◽  
Laurence Svanella-Dumas ◽  
Maria Landgraf ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Leaf Roll ◽  
Viral Population ◽  
Comprehensive Overview ◽  
Dna And Rna ◽  
Birch Trees ◽  
Viral Communities ◽  
The Impact

AbstractHigh-throughput sequencing (HTS), combined with bioinformatics for de novo discovery and assembly of plant virus or viroid genome reads, has promoted the discovery of abundant novel DNA and RNA viruses and viroids. However, the elucidation of a viral population in a single plant is rarely reported. In five birch trees of German and Finnish origin exhibiting symptoms of birch leaf-roll disease (BRLD), we identified in total five viruses, among which three are novel. The number of identified virus variants in each transcriptome ranged from one to five. The novel species are genetically - fully or partially - characterized, they belong to the genera Carlavirus, Idaeovirus and Capillovirus and they are tentatively named birch carlavirus, birch idaeovirus, and birch capillovirus, respectively. The only virus systematically detected by HTS in symptomatic trees affected by the BRLD was the recently discovered birch leafroll-associated virus. The role of the new carlavirus in BLRD etiology seems at best weak, as it was detected only in one of three symptomatic trees. Continuing studies have to clarify the impact of the carlavirus to the BLRD. The role of the Capillovirus and the Idaeovirus within the BLRD complex and whether they influence plant vitality need to be investigated. Our study reveals the viral population in single birch trees and provides a comprehensive overview for the diversities of the viral communities they harbor.

scholarly journals Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing

2021 ◽  
Author(s):  
A. Dhingra ◽  
J. Götting ◽  
P. R. Varanasi ◽  
L. Steinbrueck ◽  
S. Camiolo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Cytomegalovirus ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Stem Cell Transplant ◽  
Haematopoietic Stem Cell Transplant ◽  
Haematopoietic Stem Cell ◽  
Clinical Samples ◽  
Cell Transplant ◽  
Genotype Distribution

AbstractHuman cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.

scholarly journals Fast sequence-based microsatellite genotyping development workflow

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9085 ◽  
Author(s):  
Olivier Lepais ◽  
Emilie Chancerel ◽  
Christophe Boury ◽  
Franck Salin ◽  
Aurélie Manicki ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Ad Hoc ◽  
De Novo ◽  
Microsatellite Genotyping ◽  
Sequencing Technologies ◽  
Wide Range ◽  
Genetic Diversity And Structure ◽  
Genomic Resource ◽  
Analytical Frameworks ◽  
Different Levels

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20–40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

scholarly journals De novo Sequencing of Novel Mycoviruses From Fusarium sambucinum: An Attempt on Direct RNA Sequencing of Viral dsRNAs

2021 ◽  
Vol 12 ◽  
Author(s):  
Yukiyoshi Mizutani ◽  
Kazuma Uesaka ◽  
Ayane Ota ◽  
Matteo Calassanzio ◽  
Claudio Ratti ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Reading Ability ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Genomic Sequence ◽  
Homology Search ◽  
Fusarium Sambucinum ◽  
Consensus Sequences ◽  
Molecular Features ◽  
Wide Range

An increasing number of viruses are continuously being found in a wide range of organisms, including fungi. Recent studies have revealed a wide viral diversity in microbes and a potential importance of these viruses in the natural environment. Although virus exploration has been accelerated by short-read, high-throughput sequencing (HTS), and viral de novo sequencing is still challenging because of several biological/molecular features such as micro-diversity and secondary structure of RNA genomes. This study conducted de novo sequencing of multiple double-stranded (ds) RNA (dsRNA) elements that were obtained from fungal viruses infecting two Fusarium sambucinum strains, FA1837 and FA2242, using conventional HTS and long-read direct RNA sequencing (DRS). De novo assembly of the read data from both technologies generated near-entire genomic sequence of the viruses, and the sequence homology search and phylogenetic analysis suggested that these represented novel species of the Hypoviridae, Totiviridae, and Mitoviridae families. However, the DRS-based consensus sequences contained numerous indel errors that differed from the HTS consensus sequences, and these errors hampered accurate open reading frame (ORF) prediction. Although with its present performance, the use of DRS is premature to determine viral genome sequences, the DRS-mediated sequencing shows great potential as a user-friendly platform for a one-shot, whole-genome sequencing of RNA viruses due to its long-reading ability and relative structure-tolerant nature.

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