Sequencing viral genomes during an outbreak can facilitate response and containment efforts. In this study, we describe a reverse transcription long-range polymerase chain reaction for efficient amplification and sequencing of the Ebola virus (EBOV) genome in 2 seminested reactions. We demonstrate that our method remains robust with complex biological samples by amplifying and sequencing the EBOV genome from EBOV-infected nonhuman primates (NHPs). We further demonstrate that we are able to recover viral genomes from starting concentrations as low as 103 50% tissue culture infective dose (TCID50)/mL, suggesting that this method can be employed to sequence EBOV genomes from ecologically or clinically derived samples.
Abstract We found that environmental conditions affect the stability of severe acute respiratory syndrome coronavirus 2 in nasal mucus and sputum. The virus is more stable at low-temperature and low-humidity conditions, whereas warmer temperature and higher humidity shortened half-life. Although infectious virus was undetectable after 48 hours, viral RNA remained detectable for 7 days.
Viral load in patients with Ebola virus disease affects case fatality rate and is an important parameter used for diagnostic cutoffs, stratification in randomised controlled trials, and epidemiological studies. However, viral load in Ebola virus disease is currently estimated using numerous different assays and protocols that were not developed or validated for this purpose. Here, our aim was to conduct a laboratory-based re-evaluation of the viral loads of a large cohort of Liberian patients with Ebola virus disease and analyse these data in the broader context of the west Africa epidemic.In this retrospective observational study, whole blood samples from patients at the Eternal Love Winning Africa Ebola treatment unit (Monrovia, Liberia) were re-extracted with an optimised protocol and analysed by droplet digital PCR (ddPCR) using a novel semi-strand specific assay to measure viral load. To allow for more direct comparisons, the ddPCR viral loads were also back-calculated to cycle threshold (Ct) values. The new viral load data were then compared with the Ct values from the original diagnostic quantitative RT-PCR (qRT-PCR) testing to identify differing trends and discrepancies.Between Aug 28 and Dec 18, 2014, 727 whole blood samples from 528 individuals were collected. 463 (64%) were first-draw samples and 409 (56%) were from patients positive for Ebola virus (EBOV), species Zaire ebolavirus. Of the 307 first-draw EBOV-positive samples, 127 (41%) were from survivors and 180 (59%) were from non-survivors; 155 (50%) were women, 145 (47%) were men, and seven (2%) were not recorded, and the mean age was 29·3 (SD 15·0) years for women and 31·8 (SD 14·8) years for men. Survivors had significantly lower mean viral loads at presentation than non-survivors in both the reanalysed dataset (5·61 [95% CI 5·34-5·87] vs 7·19 [6·99-7·38] log10 EBOV RNA copies per mL; p<0·0001) and diagnostic dataset (Ct value 28·72 [27·97-29·47] vs 26·26 [25·72-26·81]; p<0·0001). However, the prognostic capacity of viral load increased with the reanalysed dataset (odds ratio [OR] of death 8·06 [95% CI 4·81-13·53], p<0·0001 for viral loads above 6·71 log10 EBOV RNA copies per mL vs OR of death 2·02 [1·27-3·20], p=0·0028 for Ct values below 27·37). Diagnostic qRT-PCR significantly (p<0·0001) underestimated viral load in both survivors and non-survivors (difference in diagnostic Ct value minus laboratory Ct value of 1·79 [95% CI 1·16-2·43] for survivors and 5·15 [4·43-5·87] for non-survivors). Six samples that were reported negative by diagnostic testing were found to be positive upon reanalysis and had high viral loads.Inaccurate viral load estimation from diagnostic Ct values is probably multifactorial; however, unaddressed PCR inhibition from tissue damage in patients with fulminant Ebola virus disease could largely account for the discrepancies observed in our study. Testing protocols for Ebola virus disease require further standardisation and validation to produce accurate viral load estimates, minimise false negatives, and allow for reliable epidemiological investigation.Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Abstract Ocular complications of Ebola virus disease are well-documented and long-term sequelae in survivors are common and lead to considerable morbidity. However, little is currently known regarding EBOV's tropism and replication kinetics within the eye. To date, limited studies have utilized in vitro infections of ocular cell lines and analyses of archived pathology samples to investigate these issues. Here, we employed ex vivo cultures of cynomolgus macaque eyes to determine the tropism of EBOV in 7 different ocular tissues: cornea, anterior sclera with bulbar conjunctiva, ciliary body, iris, lens, neural retina, and retina pigment epithelium. We report that, except for neural retina, all tissues supported EBOV replication. Retina pigment epithelium produced the fastest growth and highest viral RNA loads, although the differences were not statistically significant. Immunohistochemical staining confirmed and further characterized infection. This study demonstrates that EBOV has a broad tropism within the eye.
The unprecedented pandemic of SARS-CoV-2 has created worldwide shortages of personal protective equipment, in particular respiratory protection such as N95 respirators. SARS-CoV-2 transmission is frequently occurring in hospital settings, with numerous reported cases of nosocomial transmission highlighting the vulnerability of healthcare workers. In general, N95 respirators are designed for single use prior to disposal. Here, we have analyzed four readily available and often used decontamination methods: UV, 70% ethanol, 70C heat and vaporized hydrogen peroxide for inactivation of SARS-CoV-2 on N95 respirators. Equally important we assessed the function of the N95 respirators after multiple wear and decontamination sessions.
Diagnostics and research analyses involving samples containing maximum-containment viruses present unique challenges, and inactivation protocols compatible with downstream testing are needed. Our aim was to identify a validated viral inactivation protocol compatible with bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We assessed a panel of bacteria with 6 validated maximum-containment virus-inactivation protocols and report that inactivation with TRIzol or γ-irradiation is compatible with MALDI-TOF MS. The availability, simplicity, and rapidity of TRIzol inactivation make this method the more suitable choice.
Ambient temperature and humidity strongly affect inactivation rates of enveloped viruses, but a mechanistic, quantitative theory of these effects has been elusive. We measure the stability of SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities (RH); median estimated virus half-life is >24 hr at 10°C and 40% RH, but ∼1.5 hr at 27°C and 65% RH. Our mechanistic model uses fundamental chemistry to explain why inactivation rate increases with increased temperature and shows a U-shaped dependence on RH. The model accurately predicts existing measurements of five different human coronaviruses, suggesting that shared mechanisms may affect stability for many viruses. The results indicate scenarios of high transmission risk, point to mitigation strategies, and advance the mechanistic study of virus transmission.
Abstract Environmental conditions affect virus inactivation rate and transmission potential. Understanding those effects is critical for anticipating and mitigating epidemic spread. Ambient temperature and humidity strongly affect the inactivation rate of enveloped viruses, but a mechanistic, quantitative theory of those effects has been elusive. We measure the stability of the enveloped respiratory virus SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities; median estimated virus half-life is over 24 hours at 10 °C and 40 % RH, but approximately 1.5 hours at 27 °C and 65 % RH. Our mechanistic model uses simple chemistry to explain the increase in virus inactivation rate with increased temperature and the U-shaped dependence of inactivation rate on relative humidity. The model accurately predicts quantitative measurements from existing studies of five different human coronaviruses (including SARS-CoV-2), suggesting that shared mechanisms may determine environmental stability for many enveloped viruses. Our results indicate scenarios of particular transmission risk, point to pandemic mitigation strategies, and open new frontiers in the mechanistic study of virus transmission.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
