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.

Adipose Tissues from Human and Bat-Derived Cell Lines Support Ebola Virus Infection.

Ebola virus is a zoonotic pathogen with a geographic range covering diverse ecosystems that are home to many potential reservoir species. Although researchers have detected Ebola virus RNA and serological evidence of previous infection in different rodents and bats, the infectious virus has not been isolated. The field is missing critical knowledge about where the virus is maintained between outbreaks, either because the virus is rarely encountered, overlooked during sampling, and/or requires specific unknown conditions that regulate viral expression. This study assessed adipose tissue as a previously overlooked tissue capable of supporting Ebola virus infection. Adipose tissue is a dynamic endocrine organ helping to regulate and coordinate homeostasis, energy metabolism, and neuroendocrine and immune functions. Through in vitro infection of human and bat (Eptesicus fuscus) brown adipose tissue cultures using wild-type Ebola virus, this study showed high levels of viral replication for 28 days with no qualitative indicators of cytopathic effects. In addition, alterations in adipocyte metabolism following long-term infection were qualitatively observed through an increase in lipid droplet number while decreasing in size, a harbinger of lipolysis or adipocyte browning. The finding that bat and human adipocytes are susceptible to Ebola virus infection has important implications for potential tissue tropisms that have not yet been investigated. Additionally, the findings suggest how the metabolism of this tissue may play a role in pathogenesis, viral transmission, and/or zoonotic spillover events.

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.

A Pan-Ebolavirus Monoclonal Antibody Cocktail Provides Protection Against Ebola and Sudan Viruses.

Filoviruses, including ebolaviruses and marburgviruses, can cause severe and often fatal disease in humans. Over the past several years, antibody therapy has emerged as a promising strategy for the treatment of filovirus disease. Here, we describe 2 distinct cross-reactive monoclonal antibodies (mAbs) isolated from mice immunized with recombinant vesicular stomatitis virus-based filovirus vaccines. Both mAbs recognized the glycoproteins of multiple different ebolaviruses and exhibited broad but differential in vitro neutralization activities against these viruses. By themselves, each mAb provided partial to full protection against Ebola virus in mice, and in combination, the mAbs provided 100% protection against Sudan virus challenge in guinea pigs. This study identified novel mAbs that were elicited through immunization and able to provide protection from ebolavirus infection, thus enriching the pool of candidate therapeutics for treating Ebola disease.

Differential Infectivity of Original and Delta Variants of SARS-CoV-2 in Children Compared to Adults.

Although children of all ages are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, they have not been implicated as major drivers of transmission thus far. However, it is still unknown if this finding holds true with new variants of concern (VOC), such as Delta (B.1.617.2). This study aimed to examine differences in both viral RNA (as measured by cycle threshold [CT]) and viable-virus levels from children infected with Delta and those infected with original variants (OV). Furthermore, we aimed to compare the pediatric population infection trends to those in adults. We obtained 690 SARS-CoV-2 RT-PCR positive nasopharyngeal swabs from across Manitoba, Canada, which were further screened for mutations characteristic of VOC. Aliquots of sample were then provided for TCID50 (50% tissue culture infective dose) assays to determine infectious titers. Using a variety of statistical analyses we compared CT and infectivity of VOC in different age demographics. Comparing 122 Delta- to 175 OV-positive nasopharyngeal swab samples from children, we found that those infected with Delta are 2.7 times more likely to produce viable SARS-CoV-2 with higher titers (in TCID50 per milliliter), regardless of viral RNA levels. Moreover, comparing the pediatric samples to 130 OV- and 263 Delta-positive samples from adults, we found only that the Delta pediatric culture-positive samples had titers (TCID50 per milliliter) similar to those of culture-positive adult samples. IMPORTANCE These important findings show that children may play a larger role in viral transmission of Delta than for previously circulating SARS-CoV-2 variants. Additionally, they may suggest a mechanism for why Delta has evolved to be the predominant circulating variant.

Persistence of live virus in critically ill patients infected with SARS-COV-2: a prospective observational study.

BACKGROUND: Research on the duration of infectivity of ICU patients with COVID-19 has been sparse. Tests based on Reverse Transcriptase polymerase chain reaction (RT-PCR) detect both live virus and non-infectious viral RNA. We aimed to determine the duration of infectiousness based on viral culture of nasopharyngeal samples of patients with COVID-19. METHODS: Prospective observational study in adult intensive care units with a diagnosis of COVID-19 Pneumonia. Patients had repeated nasopharyngeal sampling performed after day 10 of ICU admission. Culture positive rate (based on viral culture on Vero cells in a level 4 lab) and Cycle threshold from RT-PCR were measured. RESULTS: Nine patients of the 108 samples (8.3%, 95% CI 3.9-15.2%) grew live virus at a median of 13 days (interquartile range 11-19) after their initial positive test. 74.1% of patients were RT-PCR positive but culture negative, and the remaining (17.6%) were RT-PCR and culture negative. Cycle threshold showed excellent ability to predict the presence of live virus, with a Ct < 25 with an AUC of 0.90 (95% CI 0.83-0.97, p < 0.001). The specificity of a Ct > 25 to predict negative viral culture was 100% (95% CI 70-100%). CONCLUSION: 8.3% of our ICU patients with COVID-19 grew live virus at a median of 13 days post-initial positive RT-PCR test. Severity of illness, use of mechanical ventilation, and time between tests did not predict the presence of live virus. Cycle threshold of > 25 had the best ability to determine the lack of live virus in these patents.

Host parameters and mode of infection influence outcome in SARS-CoV-2-infected hamsters.

The golden hamster model of SARS-CoV-2 infection recapitulates key characteristics of COVID-19. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in hamsters. We report that delivery of SARS-CoV-2 by a low- versus high-volume intranasal or intragastric route results in comparable viral titers in the lung and viral shedding. However, low-volume intranasal exposure results in milder weight loss, whereas intragastric exposure leads to a diminished capacity to regain body weight. Male hamsters, and particularly older male hamsters, display an impaired capacity to recover from illness and delayed viral clearance. These factors were found to influence the nature of the host inflammatory cytokine response but had a minimal effect on the quality and durability of the humoral immune response and susceptibility to re-infection. These data further elucidate key factors that impact pre-clinical challenge studies carried out in the hamster model of COVID-19.

SARS-CoV-2 infection and transmission in the North American deer mouse.

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown.

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.

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.

Characterization of Ebola Virus Risk to Bedside Providers in an Intensive Care Environment.

BACKGROUND: The 2014-2016 Ebola outbreak in West Africa recapitulated that nosocomial spread of Ebola virus could occur and that health care workers were at particular risk including notable cases in Europe and North America. These instances highlighted the need for centers to better prepare for potential Ebola virus cases; including understanding how the virus spreads and which interventions pose the greatest risk. METHODS: We created a fully equipped intensive care unit (ICU), within a Biosafety Level 4 (BSL4) laboratory, and infected multiple sedated non-human primates (NHPs) with Ebola virus. While providing bedside care, we sampled blood, urine, and gastric residuals; as well as buccal, ocular, nasal, rectal, and skin swabs, to assess the risks associated with routine care. We also assessed the physical environment at end-point. RESULTS: Although viral RNA was detectable in blood as early as three days post-infection, it was not detectable in the urine, gastric fluid, or swabs until late-stage disease. While droplet spread and fomite contamination were present on a few of the surfaces that were routinely touched while providing care in the ICU for the infected animal, these may have been abrogated through good routine hygiene practices. CONCLUSIONS: Overall this study has helped further our understanding of which procedures may pose the highest risk to healthcare providers and provides temporal evidence of this over the clinical course of disease.

Comparison analysis of different swabs and transport mediums suitable for SARS-CoV-2 testing following shortages.

On March 11, 2020, the World Health Organization (WHO) assessed COVID-19, caused by SARS-CoV-2, as a pandemic. As of June 1, 2020, SARS-CoV-2 has had a documented effect of over 6 million cases world-wide, amounting to over 370,000 deaths (World Health Organization, 2020. Novel Coronavirus (COVID-19) Situation. http://https://covid19.who.int/). Consequently, the high demand for testing has resulted in a depletion of commercially available consumables, including the recommended swabs and viral transport media (VTM) required for nasopharyngeal sampling. Therefore, the potential use of unvalidated alternatives must be explored to address the global shortage of testing supplies. To tackle this issue, we evaluated the utility of different swabs and transport mediums for the molecular detection of SARS-CoV-2. This study compared the performance of six swabs commonly found in primary and tertiary health care settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6" cotton tipped applicators, and HOLOGIC Aptima) for their efficacy in testing for SARS-CoV-2. Separately, the molecular detection of SARS-CoV-2 was completed from different transport mediums (DMEM, PBS, 100 % ethanol, 0.9 % normal saline and VTM), which were kept up to three days at room temperature (RT). The results indicate that there is no meaningful difference in viral yield from different swabs and most transport mediums for the collection and detection of SARS-CoV-2, indicating swab and medium alternatives could be used if supplies run out.

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.

Correction to: Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

Please note that four authors (Logan Banadyga, Alixandra Albietz, Brad Pickering, and Gary Wong) have been erroneously omitted from the author list in the published original article [1].

Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

BACKGROUND: There are currently limited data for the use of specific antiviral therapies for the treatment of Ebola virus disease (EVD). While there is anecdotal evidence that supportive care may be effective, there is a paucity of direct experimental data to demonstrate a role for supportive care in EVD. We studied the impact of ICU-level supportive care interventions including fluid resuscitation, vasoactive medications, blood transfusion, hydrocortisone, and ventilator support on the pathophysiology of EVD in rhesus macaques infected with a universally lethal dose of Ebola virus strain Makona C07. METHODS: Four NHPs were infected with a universally lethal dose Ebola virus strain Makona, in accordance with the gold standard lethal Ebola NHP challenge model. Following infection, the following therapeutic interventions were employed: continuous bedside supportive care, ventilator support, judicious fluid resuscitation, vasoactive medications, blood transfusion, and hydrocortisone as needed to treat cardiovascular compromise. A range of physiological parameters were continuously monitored to gage any response to the interventions. RESULTS: All four NHPs developed EVD and demonstrated a similar clinical course. All animals reached a terminal endpoint, which occurred at an average time of 166.5 ± 14.8 h post-infection. Fluid administration may have temporarily blunted a rise in lactate, but the effect was short lived. Vasoactive medications resulted in short-lived improvements in mean arterial pressure. Blood transfusion and hydrocortisone did not appear to have a significant positive impact on the course of the disease. CONCLUSIONS: The model employed for this study is reflective of an intramuscular infection in humans (e.g., needle stick) and is highly lethal to NHPs. Using this model, we found that the animals developed progressive severe organ dysfunction and profound shock preceding death. While the overall impact of supportive care on the observed pathophysiology was limited, we did observe some time-dependent positive responses. Since this model is highly lethal, it does not reflect the full spectrum of human EVD. Our findings support the need for continued development of animal models that replicate the spectrum of human disease as well as ongoing development of anti-Ebola therapies to complement supportive care.

Transgenic mice overexpressing desmocollin-2 (DSC2) develop cardiomyopathy associated with myocardial inflammation and fibrotic remodeling.

BACKGROUND: Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology. METHODS AND RESULTS: We generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age. CONCLUSION: Cardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy.