Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer.Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer.Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D2 reads.Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses

Eosinophilic pleural effusion due to Staphylococcus epidermidis infection: A case report

Eosinophilic pleural effusion is rare, and the cause is often obscure. A 73-year-old man with no relevant medical history presented with exertional dyspnea. Chest imaging revealed left-sided pleural effusion, and pleural fluid examination revealed eosinophilic pleural effusion. Blood tests revealed an increased peripheral blood eosinophil count and elevated Immunoglobulin E levels. Staphylococcus epidermidis was detected in pleural specimens collected via thoracoscopy. Antimicrobial therapy targeting Staphylococcus epidermidis resolved the eosinophilic pleural effusion and elevated peripheral blood eosinophil count. Staphylococcus epidermidis infection may be considered as a cause of eosinophilic pleural effusion when the diagnosis is difficult

IgG4‐related retroperitoneal fibrosis induced by nivolumab and ipilimumab in a patient with non‐small cell lung cancer: A case report

Abstract IgG4‐related diseases are adverse events that occur after receiving treatment with immune checkpoint inhibitors (ICI). This study reports the first case of IgG4‐related retroperitoneal fibrosis after the administration of chemotherapy with nivolumab and ipilimumab (NI therapy). An 80‐year‐old man developed lower abdominal pain eight months after NI therapy was initiated. Although the primary lesion maintained its reduced size on computed tomography, there was an increase in the soft tissue shadows intensity around the abdominal aorta, bladder, and seminal vesicles, suggesting retroperitoneal fibrosis. Blood tests showed elevated IgG4 levels. Computed tomography‐guided biopsy of the retroperitoneum showed B cell‐dominant lymphocyte infiltration consistent with IgG4‐related retroperitoneal fibrosis and characteristic CD8‐positive lymphocyte infiltration, suggestive of the involvement of cytotoxic T cells. Based on the clinical, imaging, and pathological findings, the patient was diagnosed with IgG4‐related retroperitoneal fibrosis due to ICI. Immunotherapy discontinuation alone did not result in improvement; therefore, steroid therapy was initiated. In clinical practice, IgG4‐related retroperitoneal fibrosis can occur as an immune‐related adverse event when administering anti‐PD‐1 and anti‐CTLA‐4 antibodies for cancer immunotherapy. Early steroid therapy could be effective in controlling this immune‐related adverse event

Histological and Transcriptomic Analysis of Adult Japanese Medaka Sampled Onboard the International Space Station

<div><p>To understand how humans adapt to the space environment, many experiments can be conducted on astronauts as they work aboard the Space Shuttle or the International Space Station (ISS). We also need animal experiments that can apply to human models and help prevent or solve the health issues we face in space travel. The Japanese medaka (<i>Oryzias latipes</i>) is a suitable model fish for studying space adaptation as evidenced by adults of the species having mated successfully in space during 15 days of flight during the second International Microgravity Laboratory mission in 1994. The eggs laid by the fish developed normally and hatched as juveniles in space. In 2012, another space experiment (“Medaka Osteoclast”) was conducted. Six-week-old male and female Japanese medaka (Cab strain osteoblast transgenic fish) were maintained in the Aquatic Habitat system for two months in the ISS. Fish of the same strain and age were used as the ground controls. Six fish were fixed with paraformaldehyde or kept in RNA stabilization reagent (n = 4) and dissected for tissue sampling after being returned to the ground, so that several principal investigators working on the project could share samples. Histology indicated no significant changes except in the ovary. However, the RNA-seq analysis of 5345 genes from six tissues revealed highly tissue-specific space responsiveness after a two-month stay in the ISS. Similar responsiveness was observed among the brain and eye, ovary and testis, and the liver and intestine. Among these six tissues, the intestine showed the highest space response with 10 genes categorized as oxidation–reduction processes (gene ontogeny term GO:0055114), and the expression levels of choriogenin precursor genes were suppressed in the ovary. Eleven genes including <i>klf9</i>, <i>klf13</i>, <i>odc1</i>, <i>hsp70</i> and <i>hif3a</i> were upregulated in more than four of the tissues examined, thus suggesting common immunoregulatory and stress responses during space adaptation.</p></div

Space responsive genes in the intestine–categorized as GO:0055114.

<p>Genes are aligned in descending order of SR values in the intestine. Acc: account, HGNC: Human Genome Organisation Gene Nomenclature Committee, RPKM: Reads Per Killobases per Million, Si: gene represented by annotated genomic sequence from the Sanger Institute, SR: space responsiveness = Log2(RPKM of SFs/RPKM of GCs), ||Z||: absolute z score, ZFIN: Zebrafish Model Organism Database</p><p>Space responsive genes in the intestine–categorized as GO:0055114.</p

Hierarchical cluster analysis of gene expression levels derived by conditional tree clustering.

<p>The top 400 genes ranked by Total Space Responsiveness (||TSR||) were analyzed using Cluster version 3.0. Red and green indicate increases and decreased expression levels of each gene in response to spaceflight, respectively.</p

GO terms for genes with more than a twofold expression in the SF intestine.

<p>The table lists the GO annotation terms for 2279 genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138799#pone.0138799.s009" target="_blank">S5 Table</a> that showed at least a twofold increase in gene expression. There were no GO annotation terms for 492 genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138799#pone.0138799.s010" target="_blank">S6 Table</a> that showed at least a twofold decrease in gene expression. The GO annotation terms show their statistically acceptable p-values (<0.05). MHC: major histocompatiblity complex, SF: spaceflight</p><p>GO terms for genes with more than a twofold expression in the SF intestine.</p

GO terms for genes differentially expressed in the SF eye.

<p>The table lists the GO annotation terms for 112 genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138799#pone.0138799.s007" target="_blank">S3 Table</a> that showed at least a twofold increase in gene expression and 84 genes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138799#pone.0138799.s008" target="_blank">S4 Table</a> that showed at least a twofold decrease in gene expression. The GO annotation terms show their statistically acceptable p-values (<0.05). SF: spaceflight</p><p>GO terms for genes differentially expressed in the SF eye.</p

Total numbers of genes and numbers of genes with at least a twofold increase or decrease in gene expression in SF tissues.

<p>Asterisks indicated p < 0.0001 as compared to the intestine by Fisher’s exact test. SF: spaceflight</p><p>Total numbers of genes and numbers of genes with at least a twofold increase or decrease in gene expression in SF tissues.</p