scholarly journals Use of Next Generation Sequencing to study two cowpox virus outbreaks

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6561 ◽  
Author(s):  
Markus H. Antwerpen ◽  
Enrico Georgi ◽  
Alexandra Nikolic ◽  
Gudrun Zoeller ◽  
Peter Wohlsein ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Group Method ◽  
Common Source ◽  
Cowpox Virus ◽  
Next Generation ◽  
Single Strain ◽  
Hemagglutinin Gene ◽  
Hospital Acquired ◽  
Virus Isolates ◽  
Generation Sequencing

BackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases.MethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.ResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.ConclusionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.

scholarly journals Use of Next Generation Sequencing to study two cowpox virus outbreaks

2018 ◽  
Author(s):  
Markus H. Antwerpen ◽  
Enrico Georgi ◽  
Alexandra Nikolic ◽  
Gudrun Zöller ◽  
Peter Wohlsein ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Cowpox Virus ◽  
Next Generation ◽  
Single Strain ◽  
Horizon 2020 ◽  
Research And Innovation ◽  
Hemagglutinin Gene ◽  
Hospital Acquired ◽  
Virus Isolates ◽  
Generation Sequencing

AbstractBackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. Sequencing of the hemagglutinin gene of various cowpox virus (CPXV) isolates resulted in an identical and unique sequence in each case pointing to a common source. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission was assumed.MethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of 4 virus isolates, 9 CPXVs from our strain collections and from DNA of 3 paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.ResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years.Aligning genomic sequences from 4 cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.DiscussionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.Funding StatementThis publication was supported by the European Virus Archive goes Global (EVAg) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 653316.This study was also supported in part by the European Union’s Horizon 2020 research and innovation program under grant agreement No 643476 (COMPARE).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

scholarly journals Rapid Metagenomic Next-Generation Sequencing during an Investigation of Hospital-Acquired Human Parainfluenza Virus 3 Infections

2016 ◽  
Vol 55 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Alexander L. Greninger ◽  
Danielle M. Zerr ◽  
Xuan Qin ◽  
Amanda L. Adler ◽  
Reigran Sampoleo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Infection Prevention ◽  
Genomic Sequence ◽  
Clinical Samples ◽  
Whole Genome ◽  
Next Generation ◽  
Sequence Coverage ◽  
Medical Unit ◽  
Hospital Acquired ◽  
Generation Sequencing

ABSTRACT Metagenomic next-generation sequencing (mNGS) is increasingly used for the unbiased detection of viruses, bacteria, fungi, and eukaryotic parasites in clinical samples. Whole-genome sequencing (WGS) of clinical bacterial isolates has been shown to inform hospital infection prevention practices, but this technology has not been utilized during potential respiratory virus outbreaks. Here, we report on the use of mNGS to inform the real-time infection prevention response to a cluster of hospital-acquired human parainfluenza 3 virus (HPIV3) infections at a children's hospital. Samples from 3 patients with hospital-acquired HPIV3 identified over a 12-day period on a general medical unit and 10 temporally associated samples from patients with community-acquired HPIV3 were analyzed. Our sample-to-sequencer time was <24 h, while our sample-to-answer turnaround time was <60 h with a hands-on time of approximately 6 h. Eight (2 cases and 6 controls) of 13 samples had sufficient sequencing coverage to yield the whole genome for HPIV3, while 10 (2 cases and 8 controls) of 13 samples gave partial genomes and all 13 samples had >1 read for HPIV3. Phylogenetic clustering revealed the presence of identical HPIV3 genomic sequence in the two of the cases with hospital-acquired infection, consistent with the concern for recent transmission within the medical unit. Adequate sequence coverage was not recovered for the third case. This work demonstrates the promise of mNGS for providing rapid information for infection prevention in addition to microbial detection.

Severe Sepsis With Septic Shock as a Consequence of a Severe Hospital-Acquired Pneumonia Resulting From Legionella Pneumophila in Children: A Case Series and Literature Review

2020 ◽  
Author(s):  
Min Ding ◽  
Chunfeng Yang ◽  
Yu-mei Li
Keyword(s):  
Septic Shock ◽  
Severe Sepsis ◽  
Next Generation Sequencing ◽  
Antibiotic Therapy ◽  
Legionella Pneumophila ◽  
High Risk Population ◽  
Next Generation ◽  
Hospital Acquired Pneumonia ◽  
Hospital Acquired ◽  
Generation Sequencing

Abstract BackgroundLegionella pneumophila is responsible for hospital or community-acquired pneumonia in adults. Immunocompromised patients with Legionella pneumophila infection are associated with rapidly severe clinical course and high mortality rates. Legionella pneumophila infection is rare in children, especially combined with extrapulmonary manifestations. In this report, we describe 2 children of severe hospital-acquired pneumonia and septic shock resulting from Legionella pneumophila. Standardized metagenomics next-generation sequencing allowed early diagnosis. Appropriate antibiotic therapy and timely extracorporeal life support were effective in achieving complete recovery. This is the first report of children with septic shock related to Legionella pneumophila infections diagnosed by metagenomics next-generation sequencing and recovered successfully.Case presentationThere were 2 cases about septic shock resulting from Legionella pneumophila. One was a six-month girl with congenital biliary atresia who underwent liver transplantation. She was suspected for rejecting the liver and admitted to hospital. The other one was a five-year-old boy with Burkitt lymphoma who was in the end of early chemotherapy. They both presented with fever, cough or shortness of breath during hospitalization. And they were transferred to PICU because of worsening dyspnea and decreased blood pressure. Patients were diagnosed by severe sepsis with septic shock likely resulting from hospital-acquired pneumonia. Metagenomics next-generation sequencing indicated L. pneumophila in blood and sputum. Aggressive intravenous fluids resuscitation and vasopressors were initiated on arrival to PICU, and they were placed on mechanical ventilation and continuous renal replacement therapy. Intravenous antibiotic therapy followed by azithromycin. Finally, the patients recovered without any long-term sequelae.ConclusionsThough sepsis or sepsis shock caused by Legionella pneumophila is rare in children, it can occur at high-risk population. Metagenomics next-generation sequencing is useful for conforming hard-to-culture pathogens and severely ill patients. The report remind pediatric physicians that we should be aware that Legionella pneumophila can cause severe sepsis or sepsis shock, especially in immunocompromised children. It is significant to select appropriate samples and pathogen detection methods in the early stage of disease.

scholarly journals Performance Characterization and Validation of Saliva as an Alternative Specimen Source for Detecting Hereditary Breast Cancer Mutations by Next Generation Sequencing

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Varsha Meghnani ◽  
Nadeem Mohammed ◽  
Christopher Giauque ◽  
Rahul Nahire ◽  
Thomas David
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Germline Mutations ◽  
Quality Parameters ◽  
Common Source ◽  
Sample Type ◽  
Next Generation ◽  
Alternative Source ◽  
Cancer Mutations ◽  
Generation Sequencing

Identification of pathogenic germline mutations by next generation sequencing is a widely accepted tool for predicting the risk of hereditary cancer development. Blood is the most common source of DNA for such tests. However, blood as a sample type has many drawbacks, including the invasive collection method, poor sample stability, and a relatively high cost of collection. Therefore, in the current study we have assessed the suitability of saliva as an alternative source of genomic DNA for the identification of germline mutations in the BRCA1/2 genes by next generation sequencing (NGS). Our results show that all of the samples yielded DNA concentrations sufficient for library preparation. The concentrations of the final libraries, which were generated by PCR using target specific primers, fall into the expected range with no notable difference between libraries generated from DNA derived from saliva or blood. Quality parameters indicate that sequencing performance is comparable across sample source. An average of (98±0.02)% variant calling concordance was obtained between the two specimen sources. Our data recommends saliva as a potential alternative for detecting germline mutation by next generation sequencing.

scholarly journals Profiling of the Causative Bacteria in Infected Lymphocysts after Lymphadenectomy for Gynecologic Cancer by Pyrosequencing the 16S Ribosomal RNA Gene Using Next-Generation Sequencing Technology

2019 ◽  
Vol 2019 ◽  
pp. 1-5 ◽  
Author(s):  
Yuya Nogami ◽  
Kouji Banno ◽  
Masataka Adachi ◽  
Haruko Kunitomi ◽  
Yusuke Kobayashi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pathogenic Bacteria ◽  
Sequence Data ◽  
Bacterial Flora ◽  
Gynecologic Cancer ◽  
False Negative ◽  
Enteric Bacteria ◽  
Next Generation ◽  
Single Strain ◽  
Generation Sequencing

Background. Surgery for gynecologic cancer with lymphadenectomy and pelvic radiotherapy can produce lymphoceles that sometimes complicate with infection, resulting in abscesses. The true pathogenic bacteria of abscesses are not always found because of false-negative results due to administered antibiotics and difficulty with detection, including for anaerobic bacteria. Analyzing bacteria flora by next-generation sequencing (NGS) using 16S ribosomal DNA may reveal the true pathogenic bacteria in abscesses. This is the first report on causative pathogens for infectious lymphocele using this technology. Methods. The subjects were patients who developed infectious lymphocele after surgery for gynecologic cancer at our hospital from July 2015 to September 2016. NGS analyses of bacterial flora were performed using specimens preserved at -80°C. Two steps of PCR were performed for purified DNA samples to obtain sequence libraries. Processing of sequence data, including operational taxonomic unit (OTU) definition, taxonomy assignment, and an OTU BLAST search were performed. All patients gave written informed consent and the study was approved by the institutional research ethics committee. Results. Six patients underwent puncture and drainage. The result in most cases indicated a single causative pathogen, including Staphylococcus lugdunensis, Streptococcus dysgalactiae, Streptococcus equinus, Enterococcus saccharolyticus, and Escherichia coli. Conclusions. NGS revealed that the causative bacteria in lymphocele infection are normally a single strain, such as a surface Gram-positive coccus or enteric bacteria. Antibiotics should be chosen as appropriate for elimination of these respective bacteria.

scholarly journals A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 70 ◽  
Author(s):  
Angela Crabtree ◽  
Emily Kizer ◽  
Samuel Hunter ◽  
James Van Leuven ◽  
Daniel New ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Next Generation Sequencing ◽  
Killer Toxin ◽  
Next Generation ◽  
High Quality ◽  
Yeast Saccharomyces Cerevisiae ◽  
Single Strain ◽  
Double Stranded Rna ◽  
Generation Sequencing ◽  
K1 Toxin

Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3′ polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.

scholarly journals Rapid metagenomic next-generation sequencing during an investigation of hospital-acquired human parainfluenza virus 3 infections

2016 ◽  
Author(s):  
Alexander L. Greninger ◽  
Danielle M Zerr ◽  
Xuan Qin ◽  
Amanda L. Adler ◽  
Janet A. Englund ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Infection Prevention ◽  
Genomic Sequence ◽  
Clinical Samples ◽  
Next Generation ◽  
Sequence Coverage ◽  
Medical Unit ◽  
Recent Transmission ◽  
Hospital Acquired ◽  
Generation Sequencing

AbstractMetagenomic next-generation sequencing (mNGS) is increasingly used for the unbiased detection of viruses, bacteria, fungi, and eukaryotic parasites in clinical samples. Whole genome sequencing (WGS) of clinical bacterial isolates has been shown to inform hospital infection prevention practices, but the use of this technology during potential respiratory virus outbreaks has not been taken advantage of. Here, we report on the use of mNGS to inform the real-time infection prevention response to a cluster of hospital-acquired human parainfluenza 3 virus (HPIV3) infections at a children’s hospital. Isolates from 3 patients with hospital-acquired HPIV3 identified over a 12-day period on a general medical unit and 10 temporally-associated isolates from patients with community-acquired of HPIV3 were analyzed. Our sample-to-sequencer time was <24 hours while our sample-to-answer turn-around time was <60 hours with a hands-on time of approximately 6 hours. Eight (2 case isolates and 6 control isolates) of 13 samples had sufficient sequencing coverage to yield whole genomes for HPIV3, while 10 (2 cases and 8 controls) of 13 samples gave partial genomes and all 13 samples had >1 read to HPIV3. Phylogenetic clustering revealed the presence of identical HPIV3 genomic sequence in the two of the cases with hospital-acquired infection, consistent with the concern for recent transmission within the medical unit. Adequate sequence coverage was not recovered for the third case. This work demonstrates the promise of mNGS to provide actionable information for infection control in addition to microbial detection.

scholarly journals Complete Genome Sequences of Two Vesicular Stomatitis Virus Isolates Collected in Mexico

2017 ◽  
Vol 5 (37) ◽  
Author(s):  
Lauro Velazquez-Salinas ◽  
Pavel Isa ◽  
Steven J. Pauszek ◽  
Luis L. Rodriguez
Keyword(s):  
New Jersey ◽  
Next Generation Sequencing ◽  
Vesicular Stomatitis Virus ◽  
Complete Genome ◽  
Full Genome ◽  
Next Generation ◽  
Genome Sequences ◽  
Vesicular Stomatitis ◽  
Virus Isolates ◽  
Generation Sequencing

ABSTRACT We report two full-genome sequences of vesicular stomatitis New Jersey virus (VSNJV) obtained by Illumina next-generation sequencing of RNA isolated from epithelial suspensions of cattle naturally infected in Mexico. These genomes represent the first full-genome sequences of vesicular stomatitis New Jersey viruses circulating in Mexico deposited in the GenBank database.

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