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Journal of Virological Methods Jan 2021Zika virus (ZIKV) infection remains a public health concern necessitating demand for long-term virus production for diagnostic assays and R&D activities. Inactivated...
Zika virus (ZIKV) infection remains a public health concern necessitating demand for long-term virus production for diagnostic assays and R&D activities. Inactivated virus constitutes an important component of the Trioplex rRT-PCR assay and serological IgM assay (MAC-ELISA). The aim of our study is to establish standard methods of ZIKV inactivation while maintaining antigenicity and RNA integrity. We tested viral supernatants by four different inactivation methods: 1. Heat inactivation at 56 °C and 60 °C; 2. Gamma-Irradiation; 3. Chemical inactivation by Beta-propiolactone (BPL) and 4. Fast-acting commercial disinfecting agents. Effectivity was measured by cytopathic effect (CPE) and plaque assay. RNA stability and antigenicity were measured by RT-PCR and MAC-ELISA, respectively. Results: Heat inactivation: Low titer samples, incubated at 56 °C for 2 h, showed neither CPE or plaques compared to high titer supernatants that required 2.5 h. Inactivation occurred at 60 °C for 60 min with all virus titers. Gamma irradiation: Samples irradiated at ≥3 Mrad for low virus concentrations and ≥5Mrad for high virus titer completely inactivated virus. Chemical Inactivation: Neither CPE nor plaques were observed with ≥0.045 % BPL inactivation of ZIKV. Disinfectant: Treatment of viral supernatants with Micro-Chem Plus™, inactivated virus in 2 min, whereas, Ethanol (70 %) and STERIS Coverage® Spray TB inactivated the virus in 5 min.
Topics: Disinfection; Humans; Indicators and Reagents; Virus Inactivation; Zika Virus; Zika Virus Infection
PubMed: 33098957
DOI: 10.1016/j.jviromet.2020.114004 -
Virus Research Nov 2021Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated...
Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. β-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.
Topics: Animals; Antigens, Viral; Antiviral Agents; COVID-19; COVID-19 Vaccines; Chlorocebus aethiops; Flocculation; Humans; Immune Sera; Propiolactone; RNA, Viral; SARS-CoV-2; Vaccines, Inactivated; Vero Cells; Virion; Virus Inactivation
PubMed: 34487766
DOI: 10.1016/j.virusres.2021.198555 -
Immunology Jan 1960The ε-toxin of Type D has been isolated in an immunologically and electrophoretically homogeneous state, by elution from DEAE cellulose. Such a preparation, containing...
The ε-toxin of Type D has been isolated in an immunologically and electrophoretically homogeneous state, by elution from DEAE cellulose. Such a preparation, containing 10,000 Lf/mg. N, sedimented as a single symmetrical peak in the ultracentrifuge, but still contained some non-protein N. The molecular weight of ε-toxin was estimated as 38,000 and its toxicity in mice as 280,000 MLD/mg. protein N. Its combination with horse antitoxin was studied by neutralization tests , by the flocculation reaction (optimum proportions), by quantitative precipitation and by double diffusion in agar gel. The immunological and chemical relationships between the non-toxic ε-precursor and the active toxin derived from it by tryptic (or other enzymic) hydrolysis, and toxoid made from either material by treatment with β-propiolactone, were also investigated.
Topics: Antitoxins; Bacterial Toxins; Clostridium perfringens; Toxins, Biological
PubMed: 14429038
DOI: No ID Found -
PloS One 2013Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of...
Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.
Topics: Animals; Anti-Bacterial Agents; Cell Line; Green Fluorescent Proteins; Humans; Inhibitory Concentration 50; Intracellular Space; Legionella pneumophila; Mice; Microbial Sensitivity Tests; Molecular Weight; Mycobacterium; Propiolactone; Species Specificity; Sulfones
PubMed: 24058631
DOI: 10.1371/journal.pone.0074813 -
Proceedings of the National Academy of... Oct 1981Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test. DNA preparations exposed to chemicals known...
Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, beta-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [gamma-32P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'-32P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyleneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10(5) DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding.
Topics: Animals; Carcinogens; Cattle; Chemical Phenomena; Chemistry; DNA; Mutagens; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Thymus Gland
PubMed: 7031643
DOI: 10.1073/pnas.78.10.6126 -
International Journal of Molecular... Jan 2020[6]-Gingerol from ginger has received considerable attention as a potential cancer therapeutic agent because of its chemopreventive and chemotherapeutic effects, as well...
[6]-Gingerol from ginger has received considerable attention as a potential cancer therapeutic agent because of its chemopreventive and chemotherapeutic effects, as well as its safety. In the current study, we examined [6]-gingerol as a natural scavenger of nine ultimate chemical carcinogens to which we are frequently exposed: glycidamide, styrene oxide, aflatoxin B1 exo-8,9-epoxide, -propiolactone, ethylene oxide, propylene oxide, 2-cyanoethylene oxide, chloroethylene oxide, and vinyl carbamate epoxide. To evaluate [6]-gingerol efficacy, we expanded our research with the examination of glutathione-the strongest natural scavenger in human cells. The corresponding activation free energies were calculated using Hartree-Fock method with three flexible basis sets and two implicit solvation models. According to our results, [6]-gingerol proves to be an extremely effective scavenger of chemical carcinogens of the epoxy type. On the other hand, with the exception of aflatoxin B1 exo-8,9-epoxide, glutathione represents a relatively poor scavenger, whose efficacy could be augmented by [6]-gingerol. Moreover, our quantum mechanical study of the alkylation reactions of chemical carcinogens with [6]-gingerol and glutathione provide valuable insights in the reaction mechanisms and the geometries of the corresponding transition states. Therefore, we strongly believe that our research forms a solid basis for further computational, experimental and clinical studies of anticarcinogenic properties of [6]-gingerol as well as for the development of novel chemoprophylactic dietary supplements. Finally, the obtained results also point to the applicability of quantum chemical methods to studies of alkylation reactions related to chemical carcinogenesis.
Topics: Aflatoxin B1; Alkylation; Anticarcinogenic Agents; Carcinogens; Catechols; Cell Line; Chemoprevention; Epoxy Compounds; Ethylene Oxide; Fatty Alcohols; Zingiber officinale; Humans; Propiolactone; Urethane
PubMed: 31973096
DOI: 10.3390/ijms21030695 -
Journal of Clinical Pathology Sep 2002alpha1 Antitrypsin was undetectable in several patient samples treated with 0.5% beta propiolactone, which was used as a virucidal agent. This study was designed to...
AIMS
alpha1 Antitrypsin was undetectable in several patient samples treated with 0.5% beta propiolactone, which was used as a virucidal agent. This study was designed to confirm beta propiolactone as the cause and determine why it might have such an effect.
METHODS
Volumes of 0, 5, 10, and 20 micro l of beta propiolactone were added to 2 ml aliquots of serum to make final concentrations of 0%, 0.25%, 0.5%, and 1% of beta propiolactone. alpha1 Antitrypsin concentrations and the pH were measured at different time intervals. The effects of adding buffer before the addition of beta propiolactone, NaOH after beta propiolactone, and 6M HCl instead of beta propiolactone were also measured.
RESULTS
The addition of beta propiolactone to a volunteer's serum showed a fall in both alpha1 antitrypsin values and pH with increasing time and concentration of beta propiolactone. This effect was also seen when adding HCl, but was partially prevented by buffering the serum or adding NaOH.
CONCLUSIONS
These results suggest that it is the acidity of the degradation products of beta propiolactone that is responsible for the fall in alpha1 antitrypsin values. This fall in alpha1 antitrypsin values was dependent on the concentration of beta propiolactone used and the length of time before the test was performed. The effect of beta propiolactone on laboratory tests should be re-evaluated, with attention being paid to sample pH, storage time, and storage temperature.
Topics: Disinfectants; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Propiolactone; Time Factors; alpha 1-Antitrypsin
PubMed: 12194994
DOI: 10.1136/jcp.55.9.659 -
Vaccine Aug 2022Inactivated viral vaccines have long been used in humans for diseases of global health threat (e.g., poliomyelitis and pandemic and seasonal influenza) and the...
Inactivated viral vaccines have long been used in humans for diseases of global health threat (e.g., poliomyelitis and pandemic and seasonal influenza) and the technology of inactivation has more recently been used for emerging diseases such as West Nile, Chikungunya, Ross River, SARS and especially for COVID-19. The Brighton Collaboration Benefit-Risk Assessment of VAccines by TechnolOgy (BRAVATO) Working Group has prepared standardized templates to describe the key considerations for the benefit and risk of several vaccine platform technologies, including inactivated viral vaccines. This paper uses the BRAVATO inactivated virus vaccine template to review the features of an inactivated whole chikungunya virus (CHIKV) vaccine that has been evaluated in several preclinical studies and clinical trials. The inactivated whole CHIKV vaccine was cultured on Vero cells and inactivated by ß-propiolactone. This provides an effective, flexible system for high-yield manufacturing. The inactivated whole CHIKV vaccine has favorable thermostability profiles, compatible with vaccine supply chains. Safety data are compiled in the current inactivated whole CHIKV vaccine safety database with unblinded data from the ongoing studies: 850 participants from phase II study (parts A and B) outside of India, and 600 participants from ongoing phase II study in India, and completed phase I clinical studies for 60 subjects. Overall, the inactivated whole CHIKV vaccine has been well tolerated, with no significant safety issues identified. Evaluation of the inactivated whole CHIKV vaccine is continuing, with 1410 participants vaccinated as of 20 April 2022. Extensive evaluation of immunogenicity in humans shows strong, durable humoral immune responses.
Topics: Animals; Antibodies, Viral; COVID-19; Chikungunya Fever; Chikungunya virus; Chlorocebus aethiops; Humans; Risk Assessment; Vaccines, Inactivated; Vero Cells; Viral Vaccines
PubMed: 35715351
DOI: 10.1016/j.vaccine.2022.06.006 -
Clinical and Experimental Vaccine... Sep 2021One of the essential goals regarding the successful control of rabies infection is the development of a safe, effective, and inexpensive vaccine. the current study aimed...
PURPOSE
One of the essential goals regarding the successful control of rabies infection is the development of a safe, effective, and inexpensive vaccine. the current study aimed to evaluate the inactivation potential of β-propiolactone (βPL), binary ethyleneimine (BEI), and hydrogen peroxide (HO).
MATERIALS AND METHODS
Estimating the inactivation kinetics of βPL, BEI, and HO revealed that the tested inactivants could completely and irreversibly inactivate rabies virus within 2, 12, and 4 hours, respectively while maintaining its viral immunogenicity. The potency of βPL, BEI, and HO inactivated vaccines was higher than the World Health Organization acceptance limit and were in the order of 3.75, 4.21, and 3.64 IU/mL, respectively. Monitoring the humoral and cellular immunity elicited post-immunization using derived hyaluronic acid (HA) and bacillus Calmette-Guérin purified protein derivative (PPD) adjuvanted rabies vaccine candidates were carried out using enzyme-linked immunosorbent assay.
RESULTS
Results demonstrated that both adjuvants could progressively enhance the release of anti-rabies total immunoglobulin G as well as the pro-inflammatory mediators (interferon-gamma and interleukin-5) relative to time. However, a higher immune response was developed in the case of HA adjuvanted rabies vaccine compared to PPD adjuvanted one. The harmful consequences of the tested adjuvants were considered via investigating the histopathological changes in the tissues of the immunized rats using hematoxylin and eosin stain. Lower adverse effects were observed post-vaccination with HA and PPD adjuvanted vaccines compared to that detected following administration of the currently used alum as standard adjuvant.
CONCLUSION
Our findings suggested that HA and PPD could serve as a promising platform for the development of newly adjuvanted rabies vaccines with elevated immune enhancing potentials and lower risk of health hazards.
PubMed: 34703805
DOI: 10.7774/cevr.2021.10.3.229 -
Journal of Clinical Microbiology Mar 2010Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to...
Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and beta-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and beta-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.
Topics: Antiviral Agents; Containment of Biohazards; Disinfection; Female; Humans; Influenza A virus; Influenza, Human; Male; Microbial Viability; Middle Aged; Neuraminidase; Occupational Exposure; Orthomyxoviridae; Virus Inactivation
PubMed: 20089763
DOI: 10.1128/JCM.02045-09