The Inability of Marburg Virus to Cause Disease in Ferrets Is Not Solely Linked to the Virus Glycoprotein.

Ebola virus (EBOV) causes lethal disease in ferrets, whereas Marburg virus (MARV) does not. To investigate this difference, we first evaluated viral entry by infecting ferret spleen cells with vesicular stomatitis viruses pseudotyped with either MARV or EBOV glycoprotein (GP). Both viruses were capable of infecting ferret spleen cells, suggesting that lack of disease is not due to a block in MARV entry. Next, we evaluated replication kinetics of authentic MARV and EBOV in ferret cell lines and demonstrated that, unlike EBOV, MARV was only capable of low levels of replication. Finally, we inoculated ferrets with a recombinant EBOV expressing MARV GP in place of EBOV GP. Infection resulted in uniformly lethal disease within 7-9 days postinfection, while MARV-inoculated animals survived until study endpoint. Together these data suggest that the inability of MARV to cause disease in ferrets is not entirely linked to GP.

Limited Benefit of Postexposure Prophylaxis With VSV-EBOV in Ebola Virus-Infected Rhesus Macaques.

Vesicular stomatitis virus-Ebola virus (VSV-EBOV) vaccine has been successfully used in ring vaccination approaches during EBOV disease outbreaks demonstrating its general benefit in short-term prophylactic vaccination, but actual proof of its benefit in true postexposure prophylaxis (PEP) for humans is missing. Animal studies have indicated PEP efficacy when VSV-EBOV was used within hours of lethal EBOV challenge. Here, we used a lower EBOV challenge dose and a combined intravenous and intramuscular VSV-EBOV administration to improve PEP efficacy in the rhesus macaque model. VSV-EBOV treatment 1 hour after EBOV challenge resulted in delayed disease progression but little benefit in outcome. Thus, we could not confirm previous results indicating questionable benefit of VSV-EBOV for EBOV PEP in a nonhuman primate model.

Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK.

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60-69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

Armstrong strain lymphocytic choriomeningitis virus infection after accidental laboratory exposure.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) is a human pathogen naturally present in wild rodents. In addition, LCMV is routinely used in immunology research as a model of viral infection in mice. The Armstrong common laboratory strain and the Clone-13 variant induce acute and chronic infections in mice, respectively. The frequent use of this virus in laboratory settings is associated with a risk of human infection for laboratory personnel. In contrast to LCMV Clone-13, few human laboratory infections with LCMV Armstrong have been reported, leading to a poor understanding of symptoms related to infection with this specific LCMV strain. CASE PRESENTATION: A researcher accidentally infected herself percutaneously with LCMV Armstrong. Symptoms including headaches, dizziness, eye pain and nausea appeared seven days post-exposure and lasted ten days. LCMV-IgM antibodies were detected at 28 days post-infection and IgG seroconversion was observed later. Complete recovery was confirmed three months post exposure. CONCLUSIONS: Research involving live viruses comes with the risk of infection for research personnel. This case is the first reported accidental human infection with LCMV Armstrong. The symptoms differed from reported infections with LCMV Clone-13, by the absence of fever and vomiting, and presence of leg numbness. This report will therefore help clinicians and public health authorities to recognize the symptoms associated with LCMV Armstrong infections and to offer appropriate counselling to individuals who accidentally expose themselves.

Role of Key Infectivity Parameters in the Transmission of Ebola Virus Makona in Macaques.

Many characteristics associated with Ebola virus disease remain to be fully understood. It is known that direct contact with infected bodily fluids is an associated risk factor, but few studies have investigated parameters associated with transmission between individuals, such as the dose of virus required to facilitate spread and route of infection. Therefore, we sought to characterize the impact by route of infection, viremia, and viral shedding through various mucosae, with regards to intraspecies transmission of Ebola virus in a nonhuman primate model. Here, challenge via the esophagus or aerosol to the face did not result in clinical disease, although seroconversion of both challenged and contact animals was observed in the latter. Subsequent intramuscular or intratracheal challenges suggest that viral loads determine transmission likelihood to naive animals in an intramuscular-challenge model, which is greatly facilitated in an intratracheal-challenge model where transmission from challenged to direct contact animal was observed consistently.

Infectivity of severe acute respiratory syndrome coronavirus 2 in children compared with adults.

BACKGROUND: The role of children in the transmission and community spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. We aimed to quantify the infectivity of SARS-CoV-2 in nasopharyngeal samples from children compared with adults. METHODS: We obtained nasopharyngeal swabs from adult and pediatric cases of coronavirus disease 2019 (COVID-19) and from their contacts who tested positive for SARS-CoV-2 in Manitoba between March and December 2020. We compared viral growth in cell culture, cycle threshold values from the reverse transcription polymerase chain reaction (RT-PCR) of the SARS-CoV-2 envelope (E) gene and the 50% tissue culture infective dose (TCID50/mL) between adults and children. RESULTS: Among 305 samples positive for SARS-CoV-2 by RT-PCR, 97 samples were from children aged 10 years or younger, 78 were from children aged 11-17 years and 130 were from adults (≥ 18 yr). Viral growth in culture was present in 31% of samples, including 18 (19%) samples from children 10 years or younger, 18 (23%) from children aged 11-17 years and 57 (44%) from adults (children v. adults, odds ratio 0.45, 95% confidence interval [CI] 0.28-0.72). The cycle threshold was 25.1 (95% CI 17.7-31.3) in children 10 years or younger, 22.2 (95% CI 18.3-29.0) in children aged 11-17 years and 18.7 (95% CI 17.9-30.4) in adults (p < 0.001). The median TCID50/mL was significantly lower in children aged 11-17 years (316, interquartile range [IQR] 178-2125) than adults (5620, IQR 1171 to 17 800, p < 0.001). Cycle threshold was an accurate predictor of positive culture in both children and adults (area under the receiver-operator curve, 0.87, 95% CI 0.81-0.93 v. 0.89, 95% CI 0.83-0.96, p = 0.6). INTERPRETATION: Compared with adults, children with nasopharyngeal swabs that tested positive for SARS-CoV-2 were less likely to grow virus in culture, and had higher cycle thresholds and lower viral concentrations, suggesting that children are not the main drivers of SARS-CoV-2 transmission.

Predicting Infectious Severe Acute Respiratory Syndrome Coronavirus 2 From Diagnostic Samples.

BACKGROUND: Reverse-transcription polymerase chain reaction (RT-PCR) has become the primary method to diagnose viral diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RT-PCR detects RNA, not infectious virus; thus, its ability to determine duration of infectivity of patients is limited. Infectivity is a critical determinant in informing public health guidelines/interventions. Our goal was to determine the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT), and infectivity in cell culture. METHODS: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR-confirmed positive samples and determined their ability to infect Vero cell lines. RESULTS: Ninety RT-PCR SARS-CoV-2-positive samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median tissue culture infectious dose/mL was 1780 (interquartile range, 282-8511). There was no growth in samples with a Ct > 24 or STT > 8 days. Multivariate logistic regression using positive viral culture as a binary predictor variable, STT, and Ct demonstrated an odds ratio (OR) for positive viral culture of 0.64 (95% confidence interval [CI], .49-.84; P < .001) for every 1-unit increase in Ct. Area under the receiver operating characteristic curve for Ct vs positive culture was OR, 0.91 (95% CI, .85-.97; P < .001), with 97% specificity obtained at a Ct of > 24. CONCLUSIONS: SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ct < 24 and STT < 8 days. Infectivity of patients with Ct > 24 and duration of symptoms > 8 days may be low. This information can inform public health policy and guide clinical, infection control, and occupational health decisions. Further studies of larger size are needed.

Hantavirus Cardiopulmonary Syndrome in Canada.

Hantavirus cardiopulmonary syndrome (HCPS) is a severe respiratory disease caused by Sin Nombre virus in North America (SNV). As of January 1, 2020, SNV has caused 143 laboratory-confirmed cases of HCPS in Canada. We review critical aspects of SNV virus epidemiology and the ecology, biology, and genetics of HCPS in Canada.

Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.

Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viraemia and abnormalities in blood count and blood chemistry were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clinical use.

Environmental Contamination and Persistence of Ebola Virus RNA in an Ebola Treatment Center.

BACKGROUND: Ebola viruses (EBOVs) are primarily transmitted by contact with infected body fluids. Ebola treatment centers (ETCs) contain areas that are exposed to body fluids through the care of patients suspected or confirmed to have EBOV disease. There are limited data documenting which areas/fomites within ETCs pose a risk for potential transmission. This study conducted environmental surveillance in 2 ETCs in Freetown, Sierra Leone, during the 2014-2016 West African Ebola outbreak. METHODS: ETCs were surveyed over a 3-week period. Sites to be swabbed were identified with input from field personnel. Swab samples were collected and tested for the presence of EBOV RNA. Ebola-positive body fluid-impregnated cotton pads were serially sampled. RESULTS: General areas of both ETCs were negative for EBOV RNA. The immediate vicinity of patients was the area most likely to be positive for EBOV RNA. Personal protective equipment became positive during patient care, but chlorine solution washes rendered them negative. CONCLUSIONS: Personal protective equipment and patient environs do become positive for EBOV RNA, but careful attention to decontamination seems to remove it. EBOV RNA was not detected in general ward spaces. Careful attention to decontamination protocols seems to be important in minimizing the presence of EBOV RNA within ETC wards.

Ebola Laboratory Response at the Eternal Love Winning Africa Campus, Monrovia, Liberia, 2014-2015.

West Africa experienced the first epidemic of Ebola virus infection, with by far the greatest number of cases in Guinea, Sierra Leone, and Liberia. The unprecedented epidemic triggered an unparalleled response, including the deployment of multiple Ebola treatment units and mobile/field diagnostic laboratories. The National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention deployed a joint laboratory to Monrovia, Liberia, in August 2014 to support the newly founded Ebola treatment unit at the Eternal Love Winning Africa (ELWA) campus. The laboratory operated initially out of a tent structure but quickly moved into a fixed-wall building owing to severe weather conditions, the need for increased security, and the high sample volume. Until May 2015, when the laboratory closed, the site handled close to 6000 clinical specimens for Ebola virus diagnosis and supported the medical staff in case patient management. Laboratory operation and safety, as well as Ebola virus diagnostic assays, are described and discussed; in addition, lessons learned for future deployments are reviewed.

Plasmodium Parasitemia Associated With Increased Survival in Ebola Virus-Infected Patients.

BACKGROUND: The ongoing Ebola outbreak in West Africa has resulted in 28 646 suspected, probable, and confirmed Ebola virus infections. Nevertheless, malaria remains a large public health burden in the region affected by the outbreak. A joint Centers for Disease Control and Prevention/National Institutes of Health diagnostic laboratory was established in Monrovia, Liberia, in August 2014, to provide laboratory diagnostics for Ebola virus. METHODS: All blood samples from suspected Ebola virus-infected patients admitted to the Médecins Sans Frontières ELWA3 Ebola treatment unit in Monrovia were tested by quantitative real-time polymerase chain reaction for the presence of Ebola virus and Plasmodium species RNA. Clinical outcome in laboratory-confirmed Ebola virus-infected patients was analyzed as a function of age, sex, Ebola viremia, and Plasmodium species parasitemia. RESULTS: The case fatality rate of 1182 patients with laboratory-confirmed Ebola virus infections was 52%. The probability of surviving decreased with increasing age and decreased with increasing Ebola viral load. Ebola virus-infected patients were 20% more likely to survive when Plasmodium species parasitemia was detected, even after controlling for Ebola viral load and age; those with the highest levels of parasitemia had a survival rate of 83%. This effect was independent of treatment with antimalarials, as this was provided to all patients. Moreover, treatment with antimalarials did not affect survival in the Ebola virus mouse model. CONCLUSIONS: Plasmodium species parasitemia is associated with an increase in the probability of surviving Ebola virus infection. More research is needed to understand the molecular mechanism underlying this remarkable phenomenon and translate it into treatment options for Ebola virus infection.

The Merits of Malaria Diagnostics during an Ebola Virus Disease Outbreak.

Malaria is a major public health concern in the countries affected by the Ebola virus disease epidemic in West Africa. We determined the feasibility of using molecular malaria diagnostics during an Ebola virus disease outbreak and report the incidence of Plasmodium spp. parasitemia in persons with suspected Ebola virus infection.

Inactivating Zaire Ebolavirus in Whole-Blood Thin Smears Used for Malaria Diagnosis.

Malaria is an important mimic or coinfection in potential Ebolavirus disease patients. Here, we evaluated the efficacy of the 100% methanol-inactivating Zaire Ebolavirus Makona variant for malaria thin-smear preparation. We determined that 100% methanol completely inactivated the virus after 15 min.

Comparaison de l'infectivité du coronavirus du syndrome respiratoire aigu sévère 2 chez les enfants et les adultes.

CONTEXTE: Le rôle des enfants dans la propagation et la transmission communautaire du coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) est encore mal compris. Nous visons à quantifier l'infectivité du SRAS-CoV-2 d'échantillons nasopharyngés provenant d'enfants comparativement à ceux provenant d'adultes. MÉTHODES: Nous avons obtenu des écouvillons nasopharyngés de cas adultes et pédiatriques de la maladie à coronavirus 2019 (COVID-19) ainsi que de leurs contacts qui ont obtenu un résultat positif à la présence du SRAS-CoV-2 lors d'un test de dépistage au Manitoba entre les mois de mars et décembre 2020. Nous avons comparé la croissance virale en culture cellulaire, les valeurs de cycle seuil de test d'amplification en chaîne par polymérase couplé à une transcription inverse (RT-PCR) de l'enveloppe (E) du gène du SRAS-CoV-2 et de la dose infectieuse pour 50 % de la culture tissulaire (DICT50/mL) entre les adultes et les enfants. RÉSULTATS: Parmi les 305 échantillons positifs à la présence du SRAS-CoV-2 validés par RT-PCR, 97 échantillons provenaient d'enfants de 10 ans et moins, 78 échantillons d'enfants de 11­17 ans et 130 échantillons d'adultes (≥ 18 ans). On a observé une croissance virale en culture dans 31 % des échantillons, dont 18 (19 %) échantillons d'enfants de 10 ans et moins, 18 (23 %) d'enfants de 11­17 ans et 57 (44 %) d'adultes (enfants c. adultes, rapport de cotes 0,45; intervalle de confiance [IC] à 95 % 0,28­0,72). Le cycle seuil était de 25,1 (IC à 95 % 17,7­31,3) chez les enfants de 10 ans et moins, 22,2 (IC à 95 % 18,3­29,0) chez les enfants de 11­17 ans et 18,7 (IC à 95 % 17,9­30,4) chez les adultes (p < 0,001). La DICT50/mL médiane était considérablement plus faible chez les enfants de 11­17 ans (316, écart interquartile [EI] 178­2125) que chez les adultes (5620, EI 1171­17 800, p < 0,001). Le cycle seuil était un indicateur exact d'une culture positive chez les enfants et les adultes (aire sous la courbe de la fonction d'efficacité du récepteur, 0,87, IC à 95 % 0,81­0,93 c. 0,89, IC à 95 % 0,83­0,96, p = 0,6). INTERPRÉTATION: Comparés aux adultes, les enfants qui ont obtenu un résultat positif à un test de dépistage du SRAS-CoV-2 à l'aide d'un écouvillon nasopharyngé étaient moins susceptibles de présenter une croissance du virus en culture et obtenaient un cycle seuil plus élevé et une concentration virale moins élevée, indiquant que les enfants ne sont pas les principaux vecteurs de la transmission du SRAS-CoV-2.

Considerations in the Use of Nonhuman Primate Models of Ebola Virus and Marburg Virus Infection.

The filoviruses, Ebola virus and Marburg virus, are zoonotic pathogens that cause severe hemorrhagic fever in humans and nonhuman primates (NHPs), with case-fatality rates ranging from 23% to 90%. The current outbreak of Ebola virus infection in West Africa, with >26 000 cases, demonstrates the long-underestimated public health danger that filoviruses pose as natural human pathogens. Currently, there are no vaccines or treatments licensed for human use. Licensure of any medical countermeasure may require demonstration of efficacy in the gold standard cynomolgus or rhesus macaque models of filovirus infection. Substantial progress has been made over the last decade in characterizing the filovirus NHP models. However, there is considerable debate over a variety of experimental conditions, including differences among filovirus isolates used, routes and doses of exposure, and euthanasia criteria, all of which may contribute to variability of results among different laboratories. As an example of the importance of understanding these differences, recent data with Ebola virus shows that an addition of a single uridine residue in the glycoprotein gene at the editing site attenuates the virus. Here, we draw on decades of experience working with filovirus-infected NHPs to provide a perspective on the importance of various experimental conditions.

A recently isolated Lassa virus from Mali demonstrates atypical clinical disease manifestations and decreased virulence in cynomolgus macaques.

The virulence of Soromba-R, a Lassa virus strain recently isolated from southern Mali, was assessed in 2 animal models of Lassa fever: inbred strain 13 guinea pigs and cynomolgus macaques. In both models, the Malian isolate demonstrated tissue tropism and viral titers similar to those of historical Lassa virus isolates from Sierra Leone (Josiah) and Liberia (Z-132); however, the Soromba-R isolate was found to be less pathogenic, as determined by decreased mortality and prolonged time to euthanasia in macaques. Interestingly, in addition to the classic indicators of Lassa fever, Soromba-R infection presented with moderate to severe pulmonary manifestations in the macaque model. Analysis of host responses demonstrated increased immune activation in Soromba-R-infected macaques, particularly in neutrophil-activating or -potentiating proinflammatory cytokines or growth factors, including tumor necrosis factor α, macrophage inflammatory protein 1α, interleukin 1ß, and granulocyte colony-stimulating factor, as well as interleukin 5, which may be responsible for the decreased lethality and uncharacteristic clinical presentation. These results suggest that the strain of Lassa virus circulating in Mali might be less pathogenic than strains circulating in the historical region of endemicity and may result in an atypical presentation for Lassa fever, which could complicate clinical diagnosis.

Mucosal Vaccination with a Newcastle Disease Virus-Vectored Vaccine Reduces Viral Loads in SARS-CoV-2-Infected Cynomolgus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.