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Aims The goal of this study was to examine, for the first time, the virulence and pathogenicity of aerosolized Burkholderia pseudomallei, strain NCTC 13392, in BALB/c mice in order to develop an animal model for testing novel medical countermeasures (MCMs) for the treatment of human acute and subacute (a disease state between acute and chronic) melioidosis. Methods and Results BALB/c mice were exposed to varying doses of aerosolized bacteria. Acute disease was seen in animals exposed to a very‐high dose (≥103 CFU per animal) and death occurred 3–4 days postchallenge (pc). Bacteria were detected in the lungs, liver, kidney and spleen. In contrast, animals exposed to a low dose (

A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures.

A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures.

Infectious SARS-CoV-2 can be recovered from the oral cavities and saliva of COVID-19 patients with potential implications for disease transmission. Reducing viral load in patient saliva using antiviral mouthwashes may therefore have a role as a control measure in limiting virus spread, particularly in dental settings. Here, the efficacy of SARS-CoV-2 inactivation by seven commercially available mouthwashes with a range of active ingredients were evaluated in vitro. We demonstrate ≥4.1 to ≥5.5 log10 reduction in SARS-CoV-2 titre following a 1 min treatment with commercially available mouthwashes containing 0.01–0.02 % stabilised hypochlorous acid or 0.58 % povidone iodine, and non-specialist mouthwashes with both alcohol-based and alcohol-free formulations designed for home use. In contrast, products containing 1.5 % hydrogen peroxide or 0.2 % chlorhexidine gluconate were ineffective against SARS-CoV-2 in these tests. This study contributes to the growing body of evidence surrounding virucidal efficacy of mouthwashes/oral rinses against SARS-CoV-2, and has important applications in reducing risk associated with aerosol generating procedures in dentistry and potentially for infection control more widely.

Infectious SARS-CoV-2 can be recovered from the oral cavities and saliva of COVID-19 patients with potential implications for disease transmission. Reducing viral load in patient saliva using antiviral mouthwashes may therefore have a role as a control measure in limiting virus spread, particularly in dental settings. Here, the efficacy of SARS-CoV-2 inactivation by seven commercially available mouthwashes with a range of active ingredients were evaluated in vitro. We demonstrate ≥4.1 to ≥5.5 log10 reduction in SARS-CoV-2 titre following a 1 min treatment with commercially available mouthwashes containing 0.01–0.02 % stabilised hypochlorous acid or 0.58 % povidone iodine, and non-specialist mouthwashes with both alcohol-based and alcohol-free formulations designed for home use. In contrast, products containing 1.5 % hydrogen peroxide or 0.2 % chlorhexidine gluconate were ineffective against SARS-CoV-2 in these tests. This study contributes to the growing body of evidence surrounding virucidal efficacy of mouthwashes/oral rinses against SARS-CoV-2, and has important applications in reducing risk associated with aerosol generating procedures in dentistry and potentially for infection control more widely.

Highlights • SARS-CoV-2 is not fully inactivated by heating to 56 °C or 60 °C for up to 60 min. • Complete inactivation occurred at 80 °C for 90 min or 95 °C for 1 or 5 min. • Heating SARS-CoV-2 to 80 °C for more than 30 min results in increased Ct values. • Significant variation in inactivation efficacy occurred between replicate samples. • Due to variability heat-inactivation protocols require local validation., The development of safe diagnostic protocols for working with SARS-CoV-2 clinical samples at Biosafety Level 2 (BSL2) requires understanding of the effect of heat-treatment on SARS-CoV-2 viability and downstream RT-PCR sensitivity. In this study heating SARS-CoV-2/England/2/2020 to 56 °C and 60 °C for 15, 30 and 60 min reduced the virus titre by between 2.1 and 4.9 log10 pfu/mL (as determined by plaque assay). Complete inactivation did not occur and there was significant variability between replicates. Viable virus was detected by plaque assay after heat-treatment at 80 °C for 15 or 30 min but not 60 or 90 min. After heat-treatment at 80 °C for 60 min infectious virus was only detected by more sensitive virus culture. No viable virus was detected after heating to 80 °C for 90 min or 95 °C for 1 or 5 min. RT-PCR sensitivity was not compromised by heating to 56 °C and 60 °C. However, RT-PCR sensitivity was reduced (≥3 Ct value increase) after heating the virus to 80 °C for 30 min or longer, or 95 °C for 1 or 5 min. In summary we found that the efficacy of heat-inactivation varies greatly depending on temperature and duration. Local validation of heat-inactivation and its effects downstream is therefore essential for molecular testing.

Infectious SARS-CoV-2 can be recovered from the oral cavities and saliva of COVID-19 patients with potential implications for disease transmission. Reducing viral load in patient saliva using antiviral mouthwashes may therefore have a role as a control measure in limiting virus spread, particularly in dental settings. Here, the efficacy of SARS-CoV-2 inactivation by seven commercially available mouthwashes with a range of active ingredients were evaluated in vitro. We demonstrate ≥4.1 to ≥5.5 log10 reduction in SARS-CoV-2 titre following a one minute treatment with commercially available mouthwashes containing 0.01-0.02% stabilised hypochlorous acid or 0.58% povidone iodine, and non-specialist mouthwashes with both alcohol-based and alcohol-free formulations designed for home use. In contrast, products containing 1.5% hydrogen peroxide or 0.2% chlorhexidine gluconate were ineffective against SARS-CoV-2 in these tests. This study contributes to the growing body of evidence surrounding virucidal efficacy of mouthwashes/oral rinses against SARS-CoV-2, and has important applications in reducing risk associated with aerosol generating procedures in dentistry and potentially for infection control more widely.

The effect of heat on SARS-CoV-2/England/2/2020 viability was assessed by plaque assay and virus culture. Heating to 56°C and 60°C for 15, 30 and 60 minutes led to a reduction in titre of between 2.1 and 4.9 log10 pfu/ml but complete inactivation was not observed. At 80°C plaques were observed after 15 and 30 minutes of heating, however after 60 minutes viable virus was only detected following virus culture. Heating to 80°C for 90 minutes and 95°C for 1 and 5 minutes resulted in no viable virus being detected. At 56°C and 60°C significant variability between replicates was observed and the titre often increased with heat-treatment time. Nucleic acids were extracted and tested by RT-PCR. Sensitivity of the RT-PCR was not compromised by heating to 56°C and 60°C. Heating to 80°C for 30 minutes or more and 95°C for 1 or 5 minutes however, resulted in an increase of at least three Ct values. This increase remained constant when different dilutions of virus underwent heat treatment. This indicates that high temperature heat inactivation of clinical samples prior to nucleic acid extraction could significantly affect the ability to detect virus in clinical samples from patients with lower viral loads by RT-PCR.

The COVID-19 pandemic has necessitated a rapid multi-faceted response by the scientific community, bringing researchers, health officials and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation., The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

The 2014 Ebola outbreak in West Africa required the rapid testing of clinical material for the presence of potentially high titre Ebola virus (EBOV). Safe, fast and effective methods for the inactivation of such clinical samples are required so that rapid diagnostic tests including downstream analysis by RT-qPCR or nucleotide sequencing can be carried out. One of the most commonly used guanidinium - based denaturing agents, AVL (Qiagen) has been shown to fully inactivate EBOV once ethanol is added, however this is not compatible with the use of automated nucleic acid extraction systems. Additional inactivation agents need to be identified that can be used in automated systems. A candidate inactivation agent is Triton X-100, a non-denaturing detergent that is frequently used in clinical nucleic acid extraction procedures and has previously been used for inactivation of EBOV. In this study the effect of 0.1% and 1.0% Triton X-100 (final concentration 0.08% and 0.8% respectively) alone and in combination with AVL on the viability of EBOV (106 TCID50/ml) spiked into commercially available pooled negative human serum was tested. The presence of viable EBOV in the treated samples was assessed by carrying out three serial passages of the samples in Vero E6 cells (37°C, 5% CO2, 1 week for each passage). At the end of each passage the cells were observed for evidence of cytopathic effect and samples were taken for rRT-PCR analysis for the presence of EBOV RNA. Before cell culture cytotoxic components of AVL and Triton X-100 were removed from the samples using size exclusion spin column technology or a hydrophobic adsorbent resin. The results of this study showed that EBOV spiked into human serum was not fully inactivated when treated with either 0.1% (v/v) Triton X-100 for 10 mins or 1.0% (v/v) Triton X-100 for 20 mins (final concentrations 0.08% and 0.8% Triton X-100 respectively). AVL alone also did not consistently provide complete inactivation. Samples treated with both AVL and 0.1% Triton X-100 for 10 or 20 mins were shown to be completely inactivated. This treatment is compatible with downstream analysis by RT-qPCR and next generation sequencing.

During the Ebola outbreak in 2014-2015 in Sierra Leone, residual clinical specimens and accompanying data were collected from routine diagnostic testing in Public Health England (PHE) led laboratories. Most of the samples with all the accompanying data were transferred to PHE laboratories in the UK for curation by PHE. The remainder have been kept securely in Sierra Leone. The biobank holds approximately 9955 samples of which 1108 tested positive for Ebola virus. Researchers from the UK and overseas, from academia, government other research organisations and commercial companies can submit proposals to the biobank to access and use the samples. The Ministry of Health and Sanitation in Sierra Leone (MOHS) retains ownership of the data and materials and is working with PHE and other researchers to develop and conduct a series of research projects that will inform future healthcare and public health strategies relating to Ebola. The Ebola Biobank Governance Group (EBGG) was established to guarantee equality of access to the biobank for the most scientifically valuable research including by researchers from low and middle-income countries. Ensuring benefit to the people of Sierra Leone is an over-arching principle for decisions of the EBGG. Four ongoing research collaborations are based on the first wave of biobank proposals approved by EBGG. Whilst the biobank is a valuable resource its completeness and sample quality are consistent with the outbreak conditions under which they were collected.

To evaluate new vaccines when human efficacy studies are not possible, the FDA's “Animal Rule” requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10 5 PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the “Animal Rule,” in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference ( P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.

Concern over the release of variola virus as an agent of bioterrorism remains high and a rapid vaccination regimen is desirable for use in the event of a confirmed release of virus. A single, high-dose (5×108 TCID50) of Bavarian Nordic's IMVAMUNE was tested in a Phase-II clinical trial, in humans, as a substitute for the standard (1×108 TCID50), using a 2-dose, 28-days apart regimen. Prior to this clinical trial taking place a Good Laboratory Practice, repeated high-dose, toxicology study was performed using IMVAMUNE, in New Zealand white rabbits and the results are reported here. Male and female rabbits were dosed twice, subcutaneously, with 5×108 TCID50 of IMVAMUNE (test) or saline (control), 7-days apart. The clinical condition, body-weight, food consumption, haematology, blood chemistry, immunogenicity, organ-weight, and macroscopic and microscopic pathology were investigated. Haematological investigations indicated changes within the white blood cell profile that were attributed to treatment with IMVAMUNE; these comprised slight increases in neutrophil and monocyte numbers, on study days 1-3 and a marginal increase in lymphocyte numbers on day 10. Macroscopic pathology revealed reddening at the sites of administration and thickened skin in IMVAMUNE, treated animals. After the second dose of IMVAMUNE 9/10 rabbits seroconverted, as detected by antibody ELISA on day 10, by day 21, 10/10 rabbits seroconverted. Treatment-related changes were not detected in other parameters. In conclusion, the subcutaneous injection of 2 high-doses of IMVAMUNE, to rabbits, was well tolerated producing only minor changes at the site of administration. Vaccinia-specific antibodies were raised in IMVAMUNE-vaccinated rabbits only.