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An imported case of monkeypox was diagnosed in December 2019 in a traveller returning from Nigeria to the UK. Subsequently, environmental sampling was performed at two adjoining single room residences occupied by the patient and their sibling. Monkeypox virus DNA was identified in multiple locations throughout both properties, and monkeypox virus was isolated from several samples three days after the patient was last in these locations. Positive samples were identified following use of both vacuum and surface sampling techniques; these methodologies allowed for environmental analysis of potentially contaminated porous and non-porous surfaces via real-time quantitative PCR analysis in addition to viral isolation to confirm the presence of infection-competent virus.This report confirms the potential for infection-competent monkeypox virus to be recovered in environmental settings associated with known positive cases and the necessity for rapid environmental assessment to reduce potential exposure to close contacts and the general public. The methods adopted in this investigation may be used for future confirmed cases of monkeypox in order to establish levels of contamination, confirm the presence of infection-competent material, and to identify locations requiring additional cleaning.Originality-Significance StatementSeveral imported cases of human monkeypox infection, an emerging infectious disease with a case fatality rate of up to 10%, have been identified in recent years including importations into the United Kingdom, the United States, Israel, and Singapore. It is likely that this phenomenon relates to decreased immunity against monkeypox infection in endemic regions that was previously provided via the smallpox vaccination programme. It is therefore likely that further imported cases of monkeypox will be reported in future; such occurrences will require significant clinical oversight, including suitable infection control measures. Environmental sampling to identify contaminated sites that may pose a risk can inform infection control guidance. This report documents an environmental sampling response following an imported case detected in late 2019 in the UK. Monkeypox virus DNA was readily identified in numerous locations throughout two domestic residences associated with the infected patient and infectious virus was isolated from several environmental samples confirming that contaminated environmental settings may pose a risk for onward transmission. The methods utilised in this report may advise future environmental responses following cases of this high consequence emerging viral disease in order to prevent secondary cases in close contacts or members of the public.

Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.

Background Understanding how severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is spread within the hospital setting is essential in order to protect staff, implement effective infection control measures, and prevent nosocomial transmission. Methods The presence of SARS-CoV-2 in the air and on environmental surfaces around hospitalized patients, with and without respiratory symptoms, was investigated. Environmental sampling was undertaken within eight hospitals in England during the first wave of the coronavirus disease 2019 outbreak. Samples were analysed using reverse transcription polymerase chain reaction (PCR) and virus isolation assays. Findings SARS-CoV-2 RNA was detected on 30 (8.9%) of 336 environmental surfaces. Cycle threshold values ranged from 28.8 to 39.1, equating to 2.2 x 105 to 59 genomic copies/swab. Concomitant bacterial counts were low, suggesting that the cleaning performed by nursing and domestic staff across all eight hospitals was effective. SARS-CoV-2 RNA was detected in four of 55 air samples taken Conclusions Effective cleaning can reduce the risk of fomite (contact) transmission, but some surface types may facilitate the survival, persistence and/or dispersal of SARS-CoV-2. The presence of low or undetectable concentrations of viral RNA in the air supports current guidance on the use of specific personal protective equipment for aerosol-generating and non-aerosol-generating procedures.

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.

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 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.

Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause outbreaks in human populations accompanied by significant mortality. Owing to the lack of approved therapies, screening programmes of potentially efficacious drugs have been undertaken. One of these studies has demonstrated the possible utility of chloroquine against EBOV using pseudotyped assays. In mouse models of EBOV disease there are conflicting reports of the therapeutic effects of chloroquine. There are currently no reports of its efficacy using the larger and more stringent guinea pig model of infection. In this study we have shown that replication of live EBOV is impaired by chloroquine in vitro. However, no protective effects were observed in vivo when EBOV-infected guinea pigs were treated with chloroquine. These results advocate that chloroquine should not be considered as a treatment strategy for EBOV.

The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting., The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT-qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log-linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource-limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats.