Bombali Ebolavirus in Mops condylurus Bats (Molossidae), Mozambique.

We detected Bombali ebolavirus RNA in 3 free-tailed bats (Mops condylurus, Molossidae) in Mozambique. Sequencing of the large protein gene revealed 98% identity with viruses previously detected in Sierra Leone, Kenya, and Guinea. Our findings further support the suspected role of Mops condylurus bats in maintaining Bombali ebolavirus.

Evaluation of viral load in patients with Ebola virus disease in Liberia: a retrospective observational study.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Effect of Recombinant Vesicular Stomatitis Virus-Zaire Ebola Virus Vaccination on Ebola Virus Disease Illness and Death, Democratic Republic of the Congo.

We conducted a retrospective cohort study to assess the effect vaccination with the live-attenuated recombinant vesicular stomatitis virus-Zaire Ebola virus vaccine had on deaths among patients who had laboratory-confirmed Ebola virus disease (EVD). We included EVD-positive patients coming to an Ebola Treatment Center in eastern Democratic Republic of the Congo during 2018-2020. Overall, 25% of patients vaccinated before symptom onset died compared with 63% of unvaccinated patients. Vaccinated patients reported fewer EVD-associated symptoms, had reduced time to clearance of viral load, and had reduced length of stay at the Ebola Treatment Center. After controlling for confounders, vaccination was strongly associated with decreased deaths. Reduction in deaths was not affected by timing of vaccination before or after EVD exposure. These findings support use of preexposure and postexposure recombinant vesicular stomatitis virus-Zaire Ebola virus vaccine as an intervention associated with improved death rates, illness, and recovery time among patients with EVD.

Evolution of Viral Pathogens Follows a Linear Order.

Although lessons have been learned from previous severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) outbreaks, the rapid evolution of the viruses means that future outbreaks of a much larger scale are possible, as shown by the current coronavirus disease 2019 (COVID-19) outbreak. Therefore, it is necessary to better understand the evolution of coronaviruses as well as viruses in general. This study reports a comparative analysis of the amino acid usage within several key viral families and genera that are prone to triggering outbreaks, including coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2], SARS-CoV, MERS-CoV, human coronavirus-HKU1 [HCoV-HKU1], HCoV-OC43, HCoV-NL63, and HCoV-229E), influenza A (H1N1 and H3N2), flavivirus (dengue virus serotypes 1 to 4 and Zika) and ebolavirus (Zaire, Sudan, and Bundibugyo ebolavirus). Our analysis reveals that the distribution of amino acid usage in the viral genome is constrained to follow a linear order, and the distribution remains closely related to the viral species within the family or genus. This constraint can be adapted to predict viral mutations and future variants of concern. By studying previous SARS and MERS outbreaks, we have adapted this naturally occurring pattern to determine that although pangolin plays a role in the outbreak of COVID-19, it may not be the sole agent as an intermediate animal. In addition to this study, our findings contribute to the understanding of viral mutations for subsequent development of vaccines and toward developing a model to determine the source of the outbreak. IMPORTANCE This study reports a comparative analysis of amino acid usage within several key viral genera that are prone to triggering outbreaks. Interestingly, there is evidence that the amino acid usage within the viral genomes is not random but in a linear order.

Optimization of Single-Dose VSV-Based COVID-19 Vaccination in Hamsters.

The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8+ T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4+ T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.

Ebola virus disease: Current perception of clinical features, diagnosis, pathogenesis, and therapeutics.

Zoonotic transmission of highly pathogenic viruses, are a cause of deadly epidemics around the globe. These are of particular concern as evident from the recent global pandemic due to Coronavirus disease 2019 (COVID-19). The genus Ebolavirus belongs to the Filoviridae family and its members are known to cause the Ebola virus disease (EVD), a highly contagious disease with a mortality rate of approximately 90%. The similarity of the clinical symptoms to those of various tropical ailments poses a high risk of misdiagnosis. Diagnostic strategies currently utilized include real time reverse transcriptase polymerase chain reaction, amongst others. No specific treatment exists at present, and the management of patients is aimed at the treatment of complications augmented with supportive clinical care. The recent outbreak of EVD in West Africa, which began in 2014, led to accelerated development of vaccines and treatment. In this review, we contemplate the origin of the ebolaviruses, discuss the clinical aspects and treatment of the disease, depict the current diagnostic strategies of the virus, as well discuss its pathogenesis. Keywords: Ebolavirus; viral origin; treatment; pathogenicity of Ebola; Ebola virus disease.

Novel modulators of p53-signaling encoded by unknown genes of emerging viruses.

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs.

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins.

Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.

Clustering and classification of virus sequence through music communication protocol and wavelet transform.

The coronavirus pandemic became a major risk in global public health. The outbreak is caused by SARS-CoV-2, a member of the coronavirus family. Though the images of the virus are familiar to us, in the present study, an attempt is made to hear the coronavirus by translating its protein spike into audio sequences. The musical features such as pitch, timbre, volume and duration are mapped based on the coronavirus protein sequence. Three different viruses Influenza, Ebola and Coronavirus were studied and compared through their auditory virus sequences by implementing Haar wavelet transform. The sonification of the coronavirus benefits in understanding the protein structures by enhancing the hidden features. Further, it makes a clear difference in the representation of coronavirus compared with other viruses, which will help in various research works related to virus sequence. This evolves as a simplified and novel way of representing the conventional computational methods.

Remdesivir targets a structurally analogous region of the Ebola virus and SARS-CoV-2 polymerases.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.