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1.
PLoS Pathog ; 18(2): e1010268, 2022 02.
Article in English | MEDLINE | ID: covidwho-1753212

ABSTRACT

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.


Subject(s)
Ebolavirus/genetics , Filoviridae Infections/virology , Filoviridae/genetics , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Genetic Complementation Test , Genome, Viral , Hemorrhagic Fever, Ebola/virology , Host Microbial Interactions , Humans , Inclusion Bodies/virology , Induced Pluripotent Stem Cells/virology , Macrophages/virology , RNA, Viral , Reverse Genetics , Vero Cells , Virion/genetics
2.
PLoS Negl Trop Dis ; 16(2): e0010205, 2022 02.
Article in English | MEDLINE | ID: covidwho-1731578

ABSTRACT

Uganda established a domestic Viral Hemorrhagic Fever (VHF) testing capacity in 2010 in response to the increasing occurrence of filovirus outbreaks. In July 2018, the neighboring Democratic Republic of Congo (DRC) experienced its 10th Ebola Virus Disease (EVD) outbreak and for the duration of the outbreak, the Ugandan Ministry of Health (MOH) initiated a national EVD preparedness stance. Almost one year later, on 10th June 2019, three family members who had contracted EVD in the DRC crossed into Uganda to seek medical treatment. Samples were collected from all the suspected cases using internationally established biosafety protocols and submitted for VHF diagnostic testing at Uganda Virus Research Institute. All samples were initially tested by RT-PCR for ebolaviruses, marburgviruses, Rift Valley fever (RVF) virus and Crimean-Congo hemorrhagic fever (CCHF) virus. Four people were identified as being positive for Zaire ebolavirus, marking the first report of Zaire ebolavirus in Uganda. In-country Next Generation Sequencing (NGS) and phylogenetic analysis was performed for the first time in Uganda, confirming the outbreak as imported from DRC at two different time point from different clades. This rapid response by the MoH, UVRI and partners led to the control of the outbreak and prevention of secondary virus transmission.


Subject(s)
Ebolavirus , Hemorrhagic Fever Virus, Crimean-Congo , Hemorrhagic Fever, Crimean , Hemorrhagic Fever, Ebola , Animals , Democratic Republic of the Congo/epidemiology , Disease Outbreaks/prevention & control , Ebolavirus/genetics , Hemorrhagic Fever, Crimean/epidemiology , Humans , Phylogeny , Uganda/epidemiology
3.
Microbiol Spectr ; 10(1): e0165521, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1673364

ABSTRACT

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.


Subject(s)
Coronavirus/genetics , Ebolavirus/genetics , Evolution, Molecular , Flavivirus/genetics , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Codon , Coronavirus/classification , Genome, Viral , Humans , Linear Models , Mutation , SARS-CoV-2/genetics , Virus Diseases/virology
4.
Front Immunol ; 12: 788235, 2021.
Article in English | MEDLINE | ID: covidwho-1650090

ABSTRACT

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.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Ebolavirus/immunology , Pandemics/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination/methods , Vesicular stomatitis Indiana virus/immunology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Ebolavirus/genetics , Female , Humans , Immunogenicity, Vaccine , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Plasmids , Spike Glycoprotein, Coronavirus/genetics , T-Lymphocytes/immunology , Treatment Outcome , Vero Cells , Vesicular stomatitis Indiana virus/genetics
5.
Acta Virol ; 65(4): 350-364, 2021.
Article in English | MEDLINE | ID: covidwho-1607905

ABSTRACT

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.


Subject(s)
COVID-19 , Ebolavirus , Hemorrhagic Fever, Ebola , Disease Outbreaks , Ebolavirus/genetics , Hemorrhagic Fever, Ebola/diagnosis , Hemorrhagic Fever, Ebola/epidemiology , Hemorrhagic Fever, Ebola/therapy , Humans , Perception , SARS-CoV-2
7.
PLoS Pathog ; 17(1): e1009033, 2021 01.
Article in English | MEDLINE | ID: covidwho-1012135

ABSTRACT

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.


Subject(s)
Communicable Diseases, Emerging/virology , RNA Viruses/metabolism , Signal Transduction/genetics , Tumor Suppressor Protein p53/metabolism , Chikungunya virus/genetics , Chikungunya virus/metabolism , Coronavirus/genetics , Coronavirus/metabolism , Ebolavirus/genetics , Ebolavirus/metabolism , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/metabolism , Humans , Influenza A virus/genetics , Influenza A virus/metabolism , Open Reading Frames , RNA Viruses/genetics , Tumor Suppressor Protein p53/genetics , Viral Nonstructural Proteins/metabolism , Zika Virus/genetics , Zika Virus/metabolism
8.
Viruses ; 12(12)2020 12 17.
Article in English | MEDLINE | ID: covidwho-979668

ABSTRACT

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.


Subject(s)
Gene Expression , Genetic Vectors/genetics , Glycoproteins/genetics , Vesicular stomatitis Indiana virus/genetics , Viral Envelope Proteins/genetics , Virus Internalization , Animals , Cell Line , Chikungunya virus/genetics , Chlorocebus aethiops , Cloning, Molecular , Ebolavirus/genetics , Gene Order , Genes, Reporter , Humans , Lassa virus/genetics , SARS-CoV-2/genetics , Time Factors , Vero Cells , Virus Replication
9.
Genomics ; 113(1 Pt 2): 778-784, 2021 01.
Article in English | MEDLINE | ID: covidwho-867194

ABSTRACT

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.


Subject(s)
Algorithms , COVID-19/virology , Genome, Viral , Music , SARS-CoV-2/classification , SARS-CoV-2/genetics , Wavelet Analysis , Amino Acid Sequence , Cluster Analysis , Coronavirus/classification , Coronavirus/genetics , Ebolavirus/classification , Ebolavirus/genetics , Humans , Middle East Respiratory Syndrome Coronavirus/classification , Middle East Respiratory Syndrome Coronavirus/genetics , Orthomyxoviridae/classification , Orthomyxoviridae/genetics , Pandemics , RNA, Viral/genetics , SARS Virus/classification , SARS Virus/genetics , Viral Proteins/genetics
10.
Proc Natl Acad Sci U S A ; 117(43): 26946-26954, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-841854

ABSTRACT

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.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Betacoronavirus/enzymology , Ebolavirus/enzymology , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Betacoronavirus/chemistry , Cell Line , Drug Tolerance/genetics , Ebolavirus/drug effects , Ebolavirus/genetics , Humans , Models, Molecular , Mutation , RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2 , Viral Nonstructural Proteins/genetics , Virus Replication/drug effects
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