Hypochlorous acid solution is a potent antiviral agent against SARS-CoV-2.

AIM: A novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suddenly appeared in Wuhan, China, and has caused pandemic. In this study, we evaluated antiviral activity of purified hypochlorous acid (HClO) against coronaviruses such as SARS-CoV-2 and transmissible gastroenteritis virus (TGEV) responsible for pig diseases. MATERIALS AND RESULTS: In a suspension test, 28.1 ppm HClO solution inactivated SARS-CoV-2 in phosphate-buffered saline with the reduction of 104 of 50% tissue culture infectious dose per ml (TCID50 per ml) within 10 s. When its concentration increased to 59.4 ppm, the virus titre decreased to below the detection limit (reduction of 5 logs TCID50 ) within 10 s even in the presence of 0.1% foetal bovine serum. In a carrier test, incubation with 125 ppm HClO solution for 10 min or 250 ppm for 5 min inactivated SARS-CoV-2 by more than 4 logs TCID50 per ml or below the detection limit. Because the titre of TGEV was 10-fold higher, TGEV was used for SARS-CoV-2 in a suspension test. As expected, 56.3 ppm HClO solution inactivated TGEV by 6 logs TCID50 within 30 s. CONCLUSIONS: In a carrier test, 125 ppm HClO solution for 10 min incubation is adequate to inactivate 4 logs TCID50 per ml of SARS-CoV-2 or more while in a suspension test 56.3 ppm HClO is adequate to inactivate 5 logs TCID50 per ml of SARS-CoV-2 when incubated for only 10 s regardless of presence or absence of organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: Effectiveness of HClO solution against SARS-CoV-2 was demonstrated by both suspension and carrier tests. HClO solution inactivated SARS-CoV-2 by 5 logs TCID50 within 10 s. HClO solution has several advantages such as none toxicity, none irritation to skin and none flammable. Thus, HClO solution can be used as a disinfectant for SARS-CoV-2.

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.

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.

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.

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.

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.

Analysis of tryptophan-rich region in Clostridium septicum alpha-toxin involved with binding to glycosylphosphatidylinositol-anchored proteins.

Clostridium septicum alpha-toxin has a unique tryptophan-rich region ((302)NGYSEWDWKWV(312)) that consists of 11 amino acid residues near the C-terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan-rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)-anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI-anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan-rich region plays an important role in the cytotoxic mechanism of alpha-toxin, especially in the binding to GPI-anchored proteins as cell receptors.

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species.

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.

Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage.

Proteolytic Clostridium botulinum type B strains were investigated for stability of toxigenicity and bont/b gene upon serial passage. Strains with bont/b gene located on their plasmids showed loss or decrease of toxigenicity during serial passage. Some strains lost the bont/b gene-encoding plasmid. The stability of the plasmids varied between strains.

Characterization of neutralizing mouse-human chimeric and shuffling antibodies against botulinum neurotoxin A.

Mouse-human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2-4, 2-5, 9-4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V(H)) and light chains (V(L)) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and mouse-human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V(H) or V(L) domains were constructed. A shuffling antibody (AC2494) that derived its V(H) and V(L) domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high-potency neutralizing chimeric antibodies by selecting and shuffling V(H) and V(L) domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.

Specificity of botulinum protease for human VAMP family proteins.

The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT.

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.

Compensatory renal growth in uninephrectomized immature rats: proliferative activity and epidermal growth factor.

Compensatory response to uninephrectomy in immature animals is stronger compared with that in adult ones and the response is due mainly to renal cell proliferation. The present study explored to show the growth pattern of the remaining kidney immediately after uninephrectomy in immature rats with special reference to proliferating activity and epidermal growth factor (EGF). Immunolocalizations of proliferating cell nuclear antigen (PCNA) and EGF in immature rat kidney were examined during the first three days after uninephrectomy. Semi-quantitative analysis of the expression of preproEGF mRNA was performed. One day after the operation, the PCNA positive cell ratios in the glomeruli and the proximal tubules were significantly higher in unilaterally nephrectomized (UNx) rats than in sham-operated (Sham) rats. In UNx and Sham rats, the proximal and distal tubular cells showed positive reactions to EGF antibody. The positive reaction of proximal tubules to EGF antibody was weaker in UNx than in Sham rats 1 day after the operation, while the degree of reactivity was not different between UNx and Sham rats 3 days after the operation. The level of expression of preproEGF mRNA in the kidney was significantly lower in UNx than in Sham rats 1 day after the operation. These results indicate that unilateral nephrectomy in immature rats causes increased proliferative activity and decreased expression of EGF in the remaining kidney during the early period of compensatory renal growth.

Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan.

The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique boNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.

Properties of corynephage attachment site and molecular epidemiology of Corynebacterium ulcerans isolated from humans and animals in Japan.

Sporadic reports of Corynebacterium ulcerans infection in humans and animals have become increasingly common throughout the world. Between 2001 and 2006, five human cases, in addition to isolation of the bacterium from the carcasses of Orcinus orca and Panthera leo, were reported in Japan. While an isolate from P. leo generated only phospholipase D (PLD), the other isolates produced both PLD and diphtheria-like toxin (DLT). Pulsed-field gel electrophoresis analysis showed that isolates from P. leo and humans were genetically homologous. Southern blotting found that a human isolate was lysogenized by two corynephages coding DLT. Sequence analysis of the region of the DLT gene revealed that the integration in C. ulcerans occurred in the same manner as that in C. diphtheriae.

Purification and sensitivity of Clostridium chauvoei hemolysin to various erythrocytes.

Using ammonium sulphate fractionation, the Clostridium chauvoei hemolysin was purified by cation exchange chromatography and sephacryl S-100 gel filtration. The molecular mass of the hemolysin, determined by SDS-PAGE was found to be approximately 27kDa. The activity of the hemolysin was determined in erythrocytes of various animals, with sensitivities observed in the order of cow, sheep, chicken, rabbit, rat, mouse, dog and horse. Temperature affected the sensitivity of erythrocytes to C. chauvoei hemolysin. These results may reflect distinct characteristics of the hemolytic activity of C. chauvoei hemolysin and that the hemolysin may be pore-forming.

Effects of maternal uninephrectomy on the development of fetal rat kidney: apoptosis and the expression of oncogenes.

The present study was designed to explore whether maternal uninephrectomy affects development of the collecting ducts in fetal kidney. Localization of DNA fragmented cells in the kidney of fetal rats from uninephrectomized mothers were examined by the terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) method. Immunohistochemistry was used to examine the localizations of bcl-2 gene products. The gene expressions for bcl-2, p53, and WT1 mRNAs were examined by using the semi-quantitative reverse transcript-polymerase chain reaction. TUNEL positive cells were more numerous in the medullary collecting ducts of the fetuses from uninephrectomized mothers than in those of the fetuses from sham-operated ones. The expressions of bcl-2, p53, and WT1 mRNAs were lower in the fetuses from uninephrectomized mothers than in the fetuses from sham-operated ones. Cells in the medullary collecting ducts showed positive reactions to anti-bcl-2 gene products antibody with the reactions being weaker in the fetuses from uninephrectomized mothers. These results showed that maternal uninephrectomy accelerated the development of fetal rat kidney and it was associated with the lowered the expression of bcl-2 in fetal rat kidney.

Protective effect of botulinum C/D mosaic toxoid against avian botulism.

Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross-reactivity. Ducks immunized once with the toxoid together with a commercial oil-adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism.