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The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Single-round infectious rotavirus, which lacks a gene essential for virion assembly, serves not only as a safe and effective rotavirus vaccine but also as an orally-administrable viral vector vaccine that induces mucosal immunity. Previously, we generated a single-round infectious rotavirus by partially deleting the viral VP6 gene, and demonstrated its potential as a promising vaccine platform. However, this system has several limitations; namely, low viral protein expression levels and safety concerns. Here, we addressed these challenges by introducing large deletions into the VP7 or VP4 genes, which are dispensable for viral protein expression but essential for virion assembly. These VP7- or VP4-defective viruses exhibited markedly higher protein expression in wild-type MA104 cells than the previously developed VP6-defective virus. In addition, the large deletions reduce the risk of viral reversion, thereby increasing both efficacy and safety. In a mouse model, these viruses induced neutralizing antibodies at levels comparable with those elicited by wild-type rotavirus, indicating their potential as rotavirus vaccines. Moreover, a VP4-defective rotavirus harboring a heterologous gene achieved high expression of heterologous proteins, warranting its application as a viral vector vaccine. To further increase safety, we established a reverse genetics system for the bovine rotavirus WC3 strain, a parental strain of the licensed live attenuated rotavirus vaccine, and successfully generated a single-round VP4-defective rotavirus based on the WC3 backbone. Taken together, these optimizations facilitate development of safe and effective single-round infectious rotavirus platforms suitable for human use.

Rotavirus, the leading cause of severe acute gastroenteritis in infants, is responsible for approximately 128,500 infant deaths globally each year. The virus’s surface proteins are highly diverse, comprising approximately 100 genotypes. This diversity affects susceptibility to neutralizing antibodies and the efficacy of vaccines. Here, we found that certain genotypes are highly resistant to serum neutralizing antibodies induced by vaccination and natural infections. Furthermore, we identified a specific region in the viral outer capsid protein that plays a significant role in determining susceptibility to neutralization. These findings may be important for predicting outbreak-causing strains and for developing more effective vaccines, ultimately contributing to the prevention of future outbreaks and improving global infant health.

Rotavirus infection is a leading cause of acute viral gastroenteritis and diarrhea in infants and young children. Owing to the limited development of effective antiviral therapies, vaccination has become the primary and most efficient strategy to reduce rotavirus-associated morbidity and mortality. Compared with classical virus attenuation strategies, reverse genetics approaches such as codon deoptimization are safer, more time-saving, more cost-effective, and more controllable. The present study describes the development of an oral live-attenuated rotavirus vaccine candidate using codon deoptimization. Based on a simian rotavirus SA11 strain, eight gene segments, encoding the structural proteins VP1, VP2, VP3, and VP6, and the non-structural proteins NSP2, NSP3, NSP4, and NSP5, were subjected to codon deoptimization. Attenuated rotavirus by multi-segment codon deoptimization (MS8cd) exhibited markedly attenuated replication both in vitro and in vivo , attributable to reduced protein production independent of mRNA stability. Despite the attenuation, MS8cd elicited robust systemic and mucosal antibody responses which were sufficient to protect neonatal mice against challenge with wild-type rotavirus in a maternal immunization model. To alter the immunogenicity, MS8cd was manipulated to encapsidate outer capsid proteins of several prevalent human rotaviruses. These reassortants exhibited altered antigenic and immunogenic properties associated with the differing genotypes of the outer capsid proteins. In conclusion, this study describes the generation of promising rotavirus vaccine candidates attenuated by codon deoptimization. They are capable of eliciting genotype-specific and broad-spectrum protective immunity against circulating strains of rotavirus. This represents a rapid-response platform for the development of novel vaccines against emerging variants.

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy 1 . Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3–q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 ( STXBP1 ). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species 2 , 3 , 4 . Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.

In a previous phase 3 study in patients with amyotrophic lateral sclerosis (ALS), edaravone did not show a significant difference in the Revised ALS Functional Rating Scale (ALSFRS-R) score compared with placebo. Post-hoc analysis of these data revealed that patients in an early stage with definite or probable diagnosis of ALS, defined by the revised El Escorial criteria, who met a select set of inclusion criteria showed a greater magnitude of effect than did the full study population. We aimed to substantiate this post-hoc result and assess safety and efficacy of edaravone in a phase 3 trial that focused on patients with early stage ALS who met the post-hoc analysis inclusion criteria. In this phase 3, randomised, double-blind, parallel-group study, patients aged 20–75 years with ALS of grade 1 or 2 in the Japan ALS Severity Classification, scores of at least 2 points on all 12 items of ALSFRS-R, forced vital capacity of 80% or more, definite or probable ALS according to the revised El Escorial criteria, and disease duration of 2 years or less were recruited from 31 hospitals in Japan. Eligible patients also had a decrease of 1–4 points in the ALSFRS-R score during a 12-week observation period before randomisation. Patients meeting all criteria were then randomly assigned 1:1 to receive 60 mg intravenous edaravone or intravenous saline placebo for 6 cycles (4 weeks per cycle with 2 weeks on, 2 weeks off) for a total treatment duration of 24 weeks. In cycle 1, the study drug or placebo was administered once per day for 14 days within a 14 day period, followed by the drug-free period. In cycle 2 and thereafter, the study drug or placebo was administered for 10 days within a 14 day period, followed by a 2 week drug-free period. Participants and investigators, including those assessing outcomes, were masked to treatment allocation. The primary efficacy outcome was the change in ALSFRS-R score from the baseline to 24 weeks (or at discontinuation if this was after the third cycle) after randomisation. The primary outcome was assessed in all patients who had received at least one treatment infusion, had at least one assessment post-baseline, and reached the end of cycle 3. For patients with missing values at the end of cycle 6, data were imputed by the last observation carried forward (LOCF) method, provided the patients had completed at least cycle 3. Safety was assessed in all patients who had received at least one treatment infusion and had at least one assessment post-baseline. This trial is registered with ClinicalTrials.gov, NCT01492686. Between Nov 28, 2011, and Sept 3, 2014, we screened 213 patients, and enrolled 192 as potential participants. Of these, 137 patients completed the observation period: 69 were randomly assigned to receive edaravone, and 68 were randomly assigned to receive placebo. 68 patients taking edaravone and 66 taking placebo were included in the primary efficacy analysis. For the primary outcome, the change in ALSFRS-R score was −5·01 (SE 0·64) in the edavarone group and −7·50 (0·66) in the placebo group. The least-squares mean difference between groups was 2·49 (SE 0·76, 95% CI 0·99–3·98; p=0·0013) in favour of edaravone. Treatment-emergent adverse events were reported in 58 (84%) patients receiving edaravone and 57 (84%) patients receiving placebo. 11 (16%) patients taking edaravone and 16 (24%) taking placebo had serious adverse events, and one (1%) patient receiving edaravone and four (6%) patients receiving placebo had adverse events (one dysphagia in edaravone group and one dyspnoea, two respiratory disorder, and one rash in the placebo group) that led to withdrawal. Edaravone showed efficacy in a small subset of people with ALS who met criteria identified in post-hoc analysis of a previous phase 3 study, showing a significantly smaller decline of ALSFRS-R score compared with placebo. There is no indication that edaravone might be effective in a wider population of patients with ALS who do not meet the criteria. Mitsubishi Tanabe Pharma Corporation.

We present a patient with Todd’s paresis who had undergone lung biopsy and who manifested increased signal intensity in the subarachnoid space of the right cerebral hemisphere on 10-h delayed enhanced fluid-attenuated inversion recovery images. We think that this was attributable to gadolinium leakage into the CSF due to increased leptomeningeal microvascular permeability in this patient with Todd’s paresis.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder that is clinically characterized by cerebellar ataxia and various associated symptoms. The disease is caused by an unstable expansion of the CAG repeat in the MJD gene. This gene is mapped to chromosome 14q32.1. To determine its genomic structure, we constructed a contig composed of six cosmid clones and eight bacterial artificial chromosome (BAC) clones. It spans approximately 300 kb and includes MJD . We also determined the complete sequence (175,330 bp) of B445M7, a human BAC clone that contains MJD . The MJD gene was found to span 48,240 bp and to contain 11 exons. Northern blot analysis showed that MJD mRNA is ubiquitously expressed in human tissues, and in at least four different sizes; namely, 1.4, 1.8, 4.5, and 7.5 kb. These different mRNA species probably result from differential splicing and polyadenylation, as shown by sequences of the 21 independent cDNA clones isolated after the screening of four human cDNA libraries prepared from whole brain, caudate, retina, and testis. The sequences of these latter clones relative to the MJD gene in B445M7 indicate that there are three alternative splicing sites and eight polyadenylation signals in MJD that are used to generate the differently sized transcripts.