Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

A Pilot Study of A2NTX, a Novel Low-Molecular-Weight Neurotoxin Derived from Subtype A2 for Post-Stroke Lower Limb Spasticity: Comparison with OnabotulinumtoxinA.

All the currently used type A botulinum neurotoxins for clinical uses are of subtype A1. We compared the efficacy and safety for the first time head-to-head between a novel botulinum toxin A2NTX prepared from subtype A2 and onabotulinumtoxinA (BOTOX) derived from A1 for post-stroke spasticity. We assessed the modified Ashworth scale (MAS) of the ankle joint, the mobility scores of Functional Independence Measure (FIM), and the grip power of the unaffected hand before and after injecting 300 units of BOTOX or A2NTX into calf muscles. The procedure was done in a blinded manner for the patient, the injecting physician, and the examiner. Stroke patients with chronic spastic hemiparesis (15 for A2NTX and 16 for BOTOX) were enrolled, and 11 for A2NTX and 13 for BOTOX (MAS of ankle; > or = 2) were entered for the MAS study. Area-under-curves of changes in MAS (primary outcome) were greater for A2NTX by day 30 (p = 0.044), and were similar by day 60. FIM was significantly improved in the A2NTX group (p = 0.005), but not in the BOTOX group by day 60. The hand grip of the unaffected limb was significantly decreased in the BOTOX-injected group (p = 0.002), but was unaffected in the A2NTX-injected group by day 60, suggesting there was less spread of A2NTX to the upper limb than there was with BOTOX. Being a small-sized pilot investigation with an imbalance in the gender of the subjects, the present study suggested superior efficacy and safety of A2NTX, and warrants a larger scale clinical trial of A2NTX to confirm these preliminary results.

Clinical Safety and Tolerability of A2NTX, a Novel Low-Molecular-Weight Neurotoxin Derived from Botulinum Neurotoxin Subtype A2, in Comparison with Subtype A1 Toxins.

All the botulinum type A neurotoxins available for clinical use are of the A1 subtype. We developed a subtype A2 low-molecular-weight (150 kD (kilo Dalton)) neurotoxin (A2NTX) with less spread and faster entry into the motor nerve terminal than A1 in vitro and in vivo. Preliminary clinical studies showed that its efficacy is superior to A1 toxins. We conducted an open study exploring its safety and tolerability profile in comparison with A1LL (LL type A1 toxin, or onabotulinumtoxinA) and a low-molecular-weight (150 kD) A1 neurotoxin (A1NTX). Those who had been using A1LL (n = 90; 50-360 mouse LD50 units) or A1NTX (n = 30; 50-580 units) were switched to A2NTX (n = 120; 25-600 units) from 2010 to 2018 (number of sessions ~27, cumulative doses ~11,640 units per patient). The adverse events for A2NTX included weakness (n = 1, ascribed to alcoholic polyneuropathy), dysphagia (1), local weakness (4), and spread to other muscles (1), whereas those for A1LL or A1NTX comprised weakness (n = 2, A1NTX), dysphagia (8), ptosis (6), local weakness (7), and spread to other muscles (15). After injections, 89 out of 120 patients preferred A2NTX to A1 for the successive sessions. The present study demonstrated that A2NTX had clinical safety up to the dose of 500 units and was well tolerated compared to A1 toxins.

Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples.

To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1-196.2 ng/ml and 0-12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.

The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin.

The necrotic enteritis toxin B-like (NetB) toxin secreted by Clostridium perfringens is a key virulence agent in the pathogenesis of avian necrotic enteritis, a disease that causes significant economic loss to the poultry industry worldwide. NetB was purified from Clostridium perfringens type G (CNEOP004) that was isolated from chickens with necrotic enteritis in Japan. EC50 of this purified NetB toward chicken liver-derived LMH cells was 0.63 µg/ml. In vivo pathogenicity of NetB to chicks produced characteristic lesions of necrotic enteritis. Analysis of the localization of the NetB monomer and oligomer molecules on LMH cells showed that both molecules of the toxin were localized in non-lipid raft regions. Moreover, removal of cholesterol with the cholesterol depletion assay carried out in LMH cells detected both oligomers and monomers of the NetB molecule. These data suggest that the NetB toxin may recognize membrane molecules different from cholesterol in non-raft region. Furthermore, NetB-binding molecules on LMH cell membranes using the toxin overlay assay with immunoblotting showed that protein molecules of different molecular sizes were bound to NetB on non-lipid raft fractions. Further studies are necessary to characterize these protein molecules to examine their specific association with NetB binding and oligomerization.

Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense.

Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B.

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 µg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 µg) and M4 (1.25 µg), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 µg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells.

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (HN and HC ) differ by 13%. The LC acts as a zinc-dependent endopeptidase, HN as the translocation domain, and HC as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, HN of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 HN is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.

Characterization of the functional activity of botulinum neurotoxin subtype B6.

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A-G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (HC ) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. HC /B1 and HC /B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its Hc .domain.

Clinical Study of New Tetravalent (Type A, B, E, and F) Botulinum Toxoid Vaccine Derived from M Toxin in Japan.

Botulinum toxin is the most poisonous substance known, and is believed to be a highly lethal as a biological weapon; researchers of the toxin are exposed to this hazard. Botulinum toxoid vaccines have been produced and used in Japan. However, since clinical studies involving these vaccines were conducted before establishment of the Ethical Guidelines for Clinical Research in Japan, their immunogenicity and safety were not systematically assessed. In this study, we produced a new tetravalent (type A, B, E, and F) botulinum toxoid vaccine, the first ever to be derived from M toxin, and conducted quality control tests with reference to the Minimum Requirements in Japan for adsorbed tetanus toxoid vaccine. Subsequently, a clinical study using the new vaccine in 48 healthy adult volunteers was conducted according to the guidelines in Japan. No clinically serious adverse event was noted. Neutralizing antibody titers for each type of toxin in the participants' sera, 1 month after the 4th injection were more than 0.25 IU/mL, indicating sufficient protection. This study demonstrated that the vaccine has marked immunogenicity and is safe for use in humans.

Development of a preliminary diagnostic measure for bovine leukosis in dairy cows using peripheral white blood cell and lymphocyte counts.

Analysis of the association between antibodies against bovine leukemia virus (BLV), BLV proviral load, and white blood cell (WBC) and lymphocyte counts was performed with 774 dairy cows. The average age, WBC counts and lymphoid cell counts tended to be higher in BLV antibody-positive cows than in antibody-negative cows. There was a similar trend in levels of proviral DNA. We analyzed age, WBC counts and lymphocyte counts by principal component analyses to create a distribution chart of the principle component scores. Using the chart, we categorized cows into four quadrants based on additional information, such as the presence of antibody and the levels of proviral DNA. Antibody-positive cows and cows with high BLV proviral load were found mostly in one quadrant of the chart, indicating that it is possible to predict the risk of infection without any knowledge on antibody status by using information, such as WBC counts as a biomarker. When only antibody-positive cows were included in the analysis, a characteristic distribution of different levels of proviral DNA was seen in the quadrants, suggesting that it is possible to estimate the extent of bovine leukosis infection by using this analysis. For this analysis and categorization of the cows into quadrants, we computed a mathematical formulation using discriminant analysis based on age and WBC and lymphocyte counts. This mathematical formulation for the hematological preliminary diagnosis of the disease is recommended as a screening tool to monitor bovine leukosis.

Genotyping and characterisation of the secretory lipolytic enzymes of Malassezia pachydermatis isolates collected from dogs.

INTRODUCTION: Malassezia species are commensals of normal skin microbial flora of humans and animals. These may become pathogenic under certain conditions such as those associated with atopic dermatitis or otitis externa in dogs. MATERIAL AND METHODS: Isolates of Malassezia pachydermatis were obtained from 27 dogs with healthy external ears and 32 dogs with otitis externa. Isolates were characterised on the basis of their first internal transcribed spacer (ITS) and internal spacer 1 (IGS1) sequences. Their extracellular lipase and phospholipase activity were also analysed. Three types of phospholipase inhibitor were used to identify the subclasses of phospholipase associated with otitis externa. RESULTS: The clinical isolates were classified into three ITS and three IGS1 sequence types. No significant differences in pathogenicity were detected among the ITS or IGS1 genotypes, and all of the isolates exhibited similar levels of lipase activity. The isolates derived from the dogs with otitis externa showed significantly higher phospholipase activity than those obtained from the dogs with healthy external ears. A phospholipase D inhibitor reduced the phospholipase activity of the isolates obtained from the dogs with otitis externa. CONCLUSIONS: This study did not show any significant differences in pathogenicity among the ITS or IGS1 genotypes but does suggest that phospholipase D might be one of the virulence factors involved in the inflammation of the external ear caused by M. pachydermatis.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia.

Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.

Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan.

Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum.

Botulinum neurotoxin type A subtype 2 confers greater safety than subtype 1 in a rat Parkinson's disease model.

Botulinum neurotoxin type A (BoNT/A) cleaves SNAP-25 and interrupts the release of acetylcholine. We previously reported that BoNT/A subtype 2 (BoNT/A2) ameliorates pathologic behavior more effectively than subtype 1 (BoNT/A1) in a rat Parkinson's disease model. Here, we further show BoNT/A2 has fewer adverse effects than BoNT/A1. We first confirmed that intrastriatal treatments of both BoNT/As had no-effect on dopaminergic terminals in the striatum. SNAP-25 cleaved by BoNT/A2 was strictly localized to the striatum on the injected side; however, SNAP-25 cleaved by BoNT/A1 diffused contralaterally. Furthermore, treatment with BoNT/A1 caused a significant reduction in body weight, while BoNT/A2 treatment did not. These results suggest that BoNT/A2 is more beneficial for clinical application against Parkinson's disease than BoNT/A1.

Botulinum neurotoxin A subtype 2 reduces pathological behaviors more effectively than subtype 1 in a rat Parkinson's disease model.

Recent reports indicate that interruption of acetylcholine release by intrastriatal injection of botulinum neurotoxin type A (BoNT/A) in a rat Parkinson's disease model reduces pathogenic behavior without adverse side effects such as memory dysfunction. Current knowledge suggests that BoNT/A subtype 1 (BoNT/A1) and BoNT/A subtype 2 (BoNT/A2) exert different effects. In the present study, we compared the effects of BoNT/A1 and BoNT/A2 on rotation behavior and in vivo cleavage of presynaptic protein SNAP-25 in a rat unilateral 6-hydroxydopamine-induced Parkinson's disease model. BoNT/A2 more effectively reduced pathogenic behavior by efficiently cleaving SNAP-25 in the striatum compared with that of BoNT/A1. Our results suggest that BoNT/A2 has greater clinical therapeutic value for treating subjects with Parkinson's disease compared to that of BoNT/A1.