Histopathological and Immunological Findings in the Common Marmoset Following Exposure to Aerosolized SARS-CoV-2.

There is an enduring requirement to develop animal models of COVID-19 to assess the efficacy of vaccines and therapeutics that can be used to treat the disease in humans. In this study, six marmosets were exposed to a small particle aerosol (1-3 µm) of SARS-CoV-2 VIC01 that delivered the virus directly to the lower respiratory tract. Following the challenge, marmosets did not develop clinical signs, although a disruption to the normal diurnal temperature rhythm was observed in three out of six animals. Early weight loss and changes to respiratory pattern and activity were also observed, yet there was limited evidence of viral replication or lung pathology associated with infection. There was a robust innate immunological response to infection, which included an early increase in circulating neutrophils and monocytes and a reduction in the proportion of circulating T-cells. Expression of the ACE2 receptor in respiratory tissues was almost absent, but there was ubiquitous expression of TMPRSS2. The results of this study indicate that exposure of marmosets to high concentrations of aerosolised SARS-CoV-2 did not result in the development of clear, reproducible signs of COVID-19.

Corrigendum: Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms.

[This corrects the article DOI: 10.3389/fcimb.2021.716436.].

Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms.

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

Investigative study into whether an insect repellent has virucidal activity against SARS-CoV-2.

A small-scale study with Mosi-guard Natural spray, an insect repellent containing Citriodiol, was performed to determine if it has virucidal activity against SARS-CoV-2. A liquid test examined the activity of the insect repellent and the individual components for virucidal activity. A surface contact test looked at the activity of the insect repellent when impregnated on a latex surface as a synthetic skin for potential topical prophylactic application. Both Mosi-guard Natural spray and Citriodiol, as well as other components of the repellent, had virucidal activity in the liquid contact test. On a latex surface used to simulate treated skin, the titre of SARS-CoV-2 was less over time on the Mosi-guard Natural-treated surface but virus was still recovered.

Survival of Lassa Virus in Blood and Tissue Culture Media and in a Small Particle Aerosol.

Knowledge of the survival and stability of a pathogen is important for understanding its risk, reducing its transmission, and establishing control measures. Lassa virus is endemic in West Africa, causes severe disease, and is an emerging pathogen of concern. Our study examined the survival of Lassa virus in blood and tissue culture media at two different temperatures. The stability of Lassa virus held within a small particle aerosol was also measured. In liquids, Lassa virus was found to decay more quickly at 30 °C compared to room temperature. Sealed samples protected from environmental desiccation were more stable than samples open to the environment. In a small particle aerosol, the decay rate of Lassa virus was determined at 2.69% per minute. This information can contribute to risk assessments and inform mitigation strategies in the event of an outbreak of Lassa virus.

Experimental aerosol survival of SARS-CoV-2 in artificial saliva and tissue culture media at medium and high humidity.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, may be transmitted via airborne droplets or contact with surfaces onto which droplets have deposited. In this study, the ability of SARS-CoV-2 to survive in the dark, at two different relative humidity values and within artificial saliva, a clinically relevant matrix, was investigated. SARS-CoV-2 was found to be stable, in the dark, in a dynamic small particle aerosol under the four experimental conditions we tested and viable virus could still be detected after 90 minutes. The decay rate and half-life was determined and decay rates ranged from 0.4 to 2.27 % per minute and the half lives ranged from 30 to 177 minutes for the different conditions. This information can be used for advice and modelling and potential mitigation strategies.

An Investigation of the Effect of Transfected Defective, Ebola Virus Genomes on Ebola Replication.

As the ongoing outbreak in the Democratic Republic of Congo illustrates, Ebola virus disease continues to pose a significant risk to humankind and this necessitates the continued development of therapeutic options. One option that warrants evaluation is that of defective genomes; these can potentially parasitize resources from the wild-type virus and may even be packaged for repeated co-infection cycles. Deletion and copy-back defective genomes have been identified and reported in the literature. As a crude, mixed preparation these were found to have limiting effects on cytopathology. Here we have used synthetic virology to clone and manufacture two deletion defective genomes. These genomes were tested with Ebola virus using in vitro cell culture and shown to inhibit viral replication; however, and against expectations, the defective genomes were not released in biologically significant numbers. We propose that EBOV might have yet unknown mechanisms to prevent parasitisation by defective interfering particles beyond the known mechanism that prevents sequential infection of the same cell. Understanding this mechanism would be necessary in any development of a defective interfering particle-based therapy.

Survival and persistence of Nipah virus in blood and tissue culture media.

Nipah virus (NiV) infection is a newly emerging zoonosis that causes severe disease in humans. Nipah virus is one of the lesser studied of the WHO emerging pathogens for which research is a priority. Survival and persistence data is important for risk management and understanding the hazard of the virus for laboratory and health care workers that may work with the virus and we present some initial findings on the survival of Nipah virus in blood and tissue culture media under different conditions. The titre of Nipah virus in blood or media at two different temperatures and exposed or sealed to the atmosphere was measured every day for three days and after a week. Nipah virus was very stable in blood in closed tubes held at room temperature with minimal decay over seven days. Decay was observed in all the other conditions tested and was more rapid in samples exposed to the atmosphere. Persistence data is useful for safety planning and risk management.

Predatory bacteria can protect SKH-1 mice from a lethal plague challenge.

With the rise of antimicrobial resistance, novel ways to treat bacterial infections are required and the use of predatory bacteria may be one such approach. Bdellovibrio species have been shown in vitro to predate on a wide range of other Gram-negative bacteria, including CDC category A/B pathogens such as Yersinia pestis. The data reported here show that treatment of SKH-1 mice with Bdellovibrio bacteriovorus HD100 provided significant protection from a lethal challenge of Yersinia pestis CO92. This is the first report of protection conferred by predation in vivo against a systemic pathogen challenge. However, this protective effect was not observed in a preliminary study with Balb/c mice. Therefore the effects of the predatory bacteria are complex and may be dependent on immune status/genetics of the host. Overall, predatory bacteria may have utility as a therapeutic modality but further work is required to understand the predator-host interaction.

Blood Coagulation Factor X Exerts Differential Effects on Adenovirus Entry into Human Lymphocytes.

It has been proposed that blood coagulation factors, principally factor X (FX), enhance the uptake of human adenovirus type 5 (Ad5) into cultured epithelial cells by bridging the viral hexon capsid protein and cell-surface heparan sulphate proteoglycans (HSPGs). We studied the effects of FX on Ad transduction of lymphoid cell lines (NK92MI, a natural killer cell line; Daudi, a B-cell line and Jurkat, a T-cell line) as well as primary peripheral blood lymphocytes (PBL) and HeLa epithelial cells using either replication-deficient Ad5, or a derivative in which the Ad5 fiber was replaced with that of another Ad type, Ad35, termed Ad5F35. PBL and NK92MI were resistant to Ad5 transduction. Transduction of Jurkat and Daudi cells by Ad5 was reduced by FX but without discernible effects on cell-surface Ad5 binding. FX reduced virus binding and transduction of all lymphoid cell lines by Ad5F35, as well as transduction of the T- and Natural Killer (NK)-cell populations of PBL. Flow cytometry analysis showed that all lymphoid cell lines were negative for HSPG components, in contrast to HeLa cells. FX reduced transduction of an HSPG-negative mutant Chinese hamster ovary cell line (CHOpgsA745) by Ad5 and Ad5F35, with Ad5F35 binding also being reduced by FX. These results point to fiber-dependent differences (Ad5 versus Ad35 fiber) in Ad binding to and transduction of human lymphoid and epithelial cells in the presence of FX.

Semliki Forest virus and Sindbis virus, but not vaccinia virus, require glycolysis for optimal replication.

Viruses are obligate intracellular pathogens which rely on the cell's machinery to produce the energy and macromolecules required for replication. Infection is associated with a modified metabolic profile and one pathway which can be modified is glycolysis. In this study, we investigated if the glycolysis pathway is required for alphavirus replication. Pre-treatment of Vero cells with three different glycolysis inhibitors (2-deoxyglucose, lonidamine and oxamate) resulted in a significant reduction (but not abrogation) of Semliki Forest virus and Sindbis virus replication, but not of the unrelated virus, vaccinia virus. Reduced virus yield was not associated with any significant cytotoxic effect and delayed treatment up to 3 h post-infection still resulted in a significant reduction. This suggested that glycolysis is required for optimal replication of alphaviruses by supporting post-entry life cycle steps.

Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition.

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.