Aerosol Survival, Disinfection and Formalin Inactivation of Nipah Virus.

Nipah virus is a relatively newly discovered emerging virus on the WHO list of priority pathogens which has the potential to cause outbreaks with high fatality rates. Whilst progress is being made in the development of animal models for evaluating vaccines and therapies, some of the more fundamental data on Nipah virus are lacking. We performed studies to generate novel information on the aerosol survival of Nipah virus and to look at the efficacy of two common disinfectants. We also performed studies to evaluate the inactivation of Nipah virus by using neutral buffered formalin. Nipah virus was relatively stable in a small particle (1-5 µm) aerosol in the dark, with it having a decay rate of 1.46%min-1. Sodium hypochlorite (at 10%) and ethanol (at 80%) reduced the titre of Nipah virus to undetectable levels. Nipah virus that was in tissue culture medium was also inactivated after 24 h in the presence of 10% formalin.

Early Isolates of SARS-CoV-2 Result in Different Pathogenesis in the Transduced Mouse Model of COVID-19.

A transduced mouse model of SARS-CoV-2 infection was established using Balb/c mice. This was achieved through the adenovirus-vectored delivery of the hACE2 gene, to render the mice transiently susceptible to the virus. The model was characterised in terms of the dissemination of hACE2 receptor expression, the dissemination of three SARS-CoV-2 virus variants in vivo up to 10 days following challenge, the resulting histopathology and the clinical signs induced in the mice. In transduced mice, the infection was short-term, with a rapid loss in body weight starting at day 2 with maximum weight loss at day 4, followed by subsequent recovery until day 10. The induced expression of the hACE2 receptor was evident in the lungs, but, upon challenge, the SARS-CoV-2 virus disseminated beyond the lungs to spleen, liver and kidney, peaking at day 2 post infection. However, by day 10 post infection, the virus was undetectable. The lung histopathology was characterised by bronchial and alveolar inflammation, which was still present at day 10 post infection. Transduced mice had differential responses to viral variants ranking CVR-Glasgow 1 > Victoria-1 > England-2 isolates in terms of body weight loss. The transduced mouse model provides a consistent and manipulatable model of SARS-CoV-2 infection to screen viral variants for their relative virulence and possible interventions.

Comparison of Aerosol Stability of Different Variants of Ebola Virus and Marburg Virus and Virulence of Aerosolised Ebola Virus in an Immune-Deficient Mouse.

During outbreaks of virus diseases, many variants may appear, some of which may be of concern. Stability in an aerosol of several Ebola virus and Marburg virus variants was investigated. Studies were performed measuring aerosol survival using the Goldberg drum but no significant difference in biological decay rates between variants was observed. In addition, historic data on virulence in a murine model of different Ebola virus variants were compared to newly presented data for Ebola virus Kikwit in the A129 Interferon alpha/beta receptor-deficient mouse model. Ebola virus Kikwit was less virulent than Ebola virus Ecran in our mouse model. The mouse model may be a useful tool for studying differences in virulence associated with different variants whereas aerosol stability studies may not need to be conducted beyond the species level.

Comparison of Experimental Middle East Respiratory Syndrome Coronavirus Infection Acquired by Three Individual Routes of Infection in the Common Marmoset.

Two strains of Middle East respiratory syndrome coronavirus (MERS-CoV), England 1 and Erasmus Medical Centre/2012 (EMC/2012), were used to challenge common marmosets (Callithrix jacchus) by three routes of infection: aerosol, oral, and intranasal. Animals challenged by the intranasal and aerosol routes presented with mild, transient disease, while those challenged by the oral route presented with a subclinical immunological response. Animals challenged with MERS-CoV strain EMC/2012 by the aerosol route responded with primary and/or secondary pyrexia. Marmosets had minimal to mild multifocal interstitial pneumonia, with the greatest relative severity being observed in animals challenged by the aerosol route. Viable virus was isolated from the host in throat swabs and lung tissue. The transient disease described is consistent with a successful host response and was characterized by the upregulation of macrophage and neutrophil function observed in all animals at the time of euthanasia. IMPORTANCE Middle East respiratory syndrome is caused by a human coronavirus, MERS-CoV, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Humans typically exhibit fever, cough, shortness of breath, gastrointestinal issues, and breathing difficulties, which can lead to pneumonia and/or renal complications. This emerging disease resulted in the first human lethal cases in 2012 and has a case fatality rate of approximately 36%. Consequently, there is a need for medical countermeasures and appropriate animal models for their assessment. This work has demonstrated the requirement for higher concentrations of virus to cause overt disease. Challenge by the aerosol, intranasal, and oral routes resulted in no or mild disease, but all animals had an immunological response. This shows that an appropriate early immunological response is able to control the disease.

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.

Quantification of Ebola virus replication kinetics in vitro.

Mathematical modelling has successfully been used to provide quantitative descriptions of many viral infections, but for the Ebola virus, which requires biosafety level 4 facilities for experimentation, modelling can play a crucial role. Ebola virus modelling efforts have primarily focused on in vivo virus kinetics, e.g., in animal models, to aid the development of antivirals and vaccines. But, thus far, these studies have not yielded a detailed specification of the infection cycle, which could provide a foundational description of the virus kinetics and thus a deeper understanding of their clinical manifestation. Here, we obtain a diverse experimental data set of the Ebola virus infection in vitro, and then make use of Bayesian inference methods to fully identify parameters in a mathematical model of the infection. Our results provide insights into the distribution of time an infected cell spends in the eclipse phase (the period between infection and the start of virus production), as well as the rate at which infectious virions lose infectivity. We suggest how these results can be used in future models to describe co-infection with defective interfering particles, which are an emerging alternative therapeutic.

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.

Haemostatic Changes in Five Patients Infected with Ebola Virus.

Knowledge on haemostatic changes in humans infected with Ebola virus is limited due to safety concerns and access to patient samples. Ethical approval was obtained to collect plasma samples from patients in Sierra Leone infected with Ebola virus over time and samples were analysed for clotting time, fibrinogen, and D-dimer levels. Plasma from healthy volunteers was also collected by two methods to determine effect of centrifugation on test results as blood collected in Sierra Leone was not centrifuged. Collecting plasma without centrifugation only affected D-dimer values. Patients with Ebola virus disease had higher PT and APTT and D-dimer values than healthy humans with plasma collected in the same manner. Fibrinogen levels in patients with Ebola virus disease were normal or lower than values measured in healthy people. Clotting times and D-dimer levels were elevated during infection with Ebola virus but return to normal over time in patients that survived and therefore could be considered prognostic. Informative data can be obtained from plasma collected without centrifugation which could improve patient monitoring in hazardous environments.

ICTV Virus Taxonomy Profile: Filoviridae.

Members of the family Filoviridae produce variously shaped, often filamentous, enveloped virions containing linear non-segmented, negative-sense RNA genomes of 15-19 kb. Several filoviruses (e.g., Ebola virus) are pathogenic for humans and are highly virulent. Several filoviruses infect bats (e.g., Marburg virus), whereas the hosts of most other filoviruses are unknown. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on Filoviridae, which is available at www.ictv.global/report/filoviridae.

Two-Center Evaluation of Disinfectant Efficacy against Ebola Virus in Clinical and Laboratory Matrices.

Ebola virus (EBOV) in body fluids poses risk for virus transmission. However, there are limited experimental data for such matrices on the disinfectant efficacy against EBOV. We evaluated the effectiveness of disinfectants against EBOV in blood on surfaces. Only 5% peracetic acid consistently reduced EBOV titers in dried blood to the assay limit of quantification.

Implementation of Objective PASC-Derived Taxon Demarcation Criteria for Official Classification of Filoviruses.

The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values (≈50% for genera, ≈30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55-58% sequence diversity threshold range for genera and at the 23-36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel "filovirus." Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences.

Ebola Virus Makona Shows Reduced Lethality in an Immune-deficient Mouse Model.

Ebola virus Makona (EBOV-Makona; from the 2013-2016 West Africa outbreak) shows decreased virulence in an immune-deficient mouse model, compared with a strain from 1976. Unlike other filoviruses tested, EBOV-Makona may be slightly more virulent by the aerosol route than by the injected route, as 2 mice died following aerosol exposure, compared with no mortality among mice that received intraperitoneal injection of equivalent or higher doses. Although most mice did not succumb to infection, the detection of an immunoglobulin G antibody response along with observed clinical signs suggest that the mice were infected but able to clear the infection and recover. We hypothesize that this may be due to the growth rates and kinetics of the virus, which appear slower than that for other filoviruses and consequently give more time for an immune response that results in clearance of the virus. In this instance, the immune-deficient mouse model is unlikely to be appropriate for testing medical countermeasures against this EBOV-Makona stock but may provide insight into pathogenesis and the immune response to virus.

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.

Experimental Respiratory Infection of Marmosets (Callithrix jacchus) With Ebola Virus Kikwit.

Ebola virus (EBOV) causes a highly infectious and lethal hemorrhagic fever in primates with high fatality rates during outbreaks and EBOV may be exploited as a potential biothreat pathogen. There is therefore a need to develop and license appropriate medical countermeasures against this virus. To determine whether the common marmoset (Callithrix jacchus) would be an appropriate model to assess vaccines or therapies against EBOV disease (EVD), initial susceptibility, lethality and pathogenesis studies were performed. Low doses of EBOV-Kikwit, between 4 and 27 times the 50% tissue culture infectious dose, were sufficient to cause a lethal, reproducible infection. Animals became febrile between days 5 and 6, maintaining a high fever before succumbing to EVD between 6 and 8 days after challenge. Typical signs of EVD were observed. Pathogenesis studies revealed that virus was isolated from the lungs of animals beginning on day 3 after challenge and from the liver, spleen and blood beginning on day 5. The most striking features were observed in animals that succumbed to infection, including high viral titers in all organs, increased levels of liver function enzymes and blood clotting times, decreased levels of platelets, multifocal moderate to severe hepatitis, and perivascular edema.

Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus).

Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10(8) cfu Burkholderia pseudomallei within 22-85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10(2) cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non-necrotic multifocal solid lesions in the livers and spleens and multi-organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10(2) cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non-necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.