RNAウイルスに対する迅速な全ゲノム決定法の構築

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5′ and 3′ terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences’ accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.権利情報：© 2023 Misu, Yoshikawa, Sugimoto, Takamatsu, Kurosu, Ouji, Yoshikawa, Shimojima, Ebihara and Saijo. This is an open-access article distributed under　the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is　permitted which does not comply with these terms

Controlling oxygen coordination and valence of network forming cations

Understanding the structure-property relationship of glass material is still challenging due to a lack of periodicity in disordered materials. Here, we report the properties and atomic structure of vanadium phosphate glasses characterized by reverse Monte Carlo modelling based on neutron/synchrotron X-ray diffraction and EXAFS data, supplemented by Raman and NMR spectroscopy. In vanadium-rich glass, the water durability, thermal stability and hardness improve as the amount of P2O5 increases, and the network former of the glass changes from VOx polyhedra to the interplay between VOx polyhedra and PO4 tetrahedra. We find for the first time that the coordination number of oxygen atoms around a V4+ is four, which is an unusually small coordination number, and plays an important role for water durability, thermal stability and hardness. Furthermore, we show that the similarity between glass and crystal beyond the nearest neighbour distance is important for glass properties. These results demonstrate that controlling the oxygen coordination and valence of the network-forming cation is necessary for designing the properties of glass

Postoperative Radiographic Early-Onset Adjacent Segment Degeneration after Single-Level L4–L5 Posterior Lumbar Interbody Fusion in Patients without Preoperative Severe Sagittal Spinal Imbalance

Study Design Retrospective study. Purpose To investigate the relationship between preoperative total spinal sagittal alignment and the early onset of adjacent segment degeneration (ASD) after single-level posterior lumbar interbody fusion (PLIF) in patients with normal sagittal spinal alignment. Overview of Literature Postoperative early-onset ASD is one of the complications after L4–L5 PLIF, a common surgical procedure for lumbar degenerative disease in patents without severe sagittal imbalance. A better understanding of the preoperative characteristics of total spinal sagittal alignment associated with early-onset ASD could help prevent the condition. Methods The study included 70 consecutive patients diagnosed with lumbar degenerative disease who underwent single-level L4–L5 PLIF between 2011 and 2015. They were divided into two groups based on the radiographic progression of L3–L4 degeneration after 1-year follow-up: the ASD and the non-ASD (NASD) group. The following radiographic parameters were preoperatively and postoperatively measured: sagittal vertebral axis (SVA), thoracic kyphosis (TK), lumbar lordosis, pelvic tilt, and pelvic incidence (PI). Results Eight of the 70 patients (11%) experienced ASD after PLIF (three males and five females; age, 64.4±7.7 years). The NASD group comprised 20 males and 42 females (age, 67.7±9.3 years). Six patients of the ASD group showed decreased L3–L4 disc height, one had L3–L4 local kyphosis, and one showed both changes. Preoperative SVA, PI, and TK were significantly smaller in the ASD group than in the NASD group (p <0.05). Conclusions A preoperative small SVA and TK with small PI were the characteristic alignments for the risk of early-onset ASD in patients without preoperative severe sagittal spinal imbalance undergoing L4–L5 single-level PLIF

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection

Isolation of dengue serotype 3 virus from the cerebrospinal fluid of an encephalitis patient in Hai Phong, Vietnam in 2013

Dengue encephalitis (DE) is characterized as unusual presentation of dengue infection. Despite the reports that DE accounts for only 1-5% of dengue cases, this disease tends to be increasingly reported to threaten global human health throughout dengue endemic areas particularly in Southeast Asia. The molecular information of clinically characterized, neurotropic dengue virus (DENV) in human beings is extremely scarce despite it playing an important role in deciphering the pathogenesis of dengue-related neurological cases. Here we report a case of DE caused by DENV3 genotype III in a male patient with atypical symptoms of DENV infection in Hai Phong, Vietnam in 2013. The virus isolated from the cerebrospinal fluid of this case-patient was closely related to DENV3 genotype III strains isolated from serum of two other patients, who manifested classical dengue in the same year and residing in the same area as the case-patient. It is noteworthy to mention that in 2013, DENV3 genotype III was detected for the first time in Vietnam

Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization

The mobilization of hematopoietic stem cells does not require osteoclasts, which may even have an inhibitory effect