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1.
Viruses ; 14(4)2022 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-35458510

RESUMO

During outbreaks of virus diseases, many variants may appear, some of which may be of concern. Stability in an aerosol of several Ebola virus and Marburg virus variants was investigated. Studies were performed measuring aerosol survival using the Goldberg drum but no significant difference in biological decay rates between variants was observed. In addition, historic data on virulence in a murine model of different Ebola virus variants were compared to newly presented data for Ebola virus Kikwit in the A129 Interferon alpha/beta receptor-deficient mouse model. Ebola virus Kikwit was less virulent than Ebola virus Ecran in our mouse model. The mouse model may be a useful tool for studying differences in virulence associated with different variants whereas aerosol stability studies may not need to be conducted beyond the species level.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Doença do Vírus de Marburg , Marburgvirus , Aerossóis , Animais , Modelos Animais de Doenças , Ebolavirus/genética , Camundongos , Virulência
2.
Virulence ; 13(1): 609-633, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35363588

RESUMO

Marburg virus (MARV) has been a major concern since 1967, with two major outbreaks occurring in 1998 and 2004. Infection from MARV results in severe hemorrhagic fever, causing organ dysfunction and death. Exposure to fruit bats in caves and mines, and human-to-human transmission had major roles in the amplification of MARV outbreaks in African countries. The high fatality rate of up to 90% demands the broad study of MARV diseases (MVD) that correspond with MARV infection. Since large outbreaks are rare for MARV, clinical investigations are often inadequate for providing the substantial data necessary to determine the treatment of MARV disease. Therefore, an overall review may contribute to minimizing the limitations associated with future medical research and improve the clinical management of MVD. In this review, we sought to analyze and amalgamate significant information regarding MARV disease epidemics, pathophysiology, and management approaches to provide a better understanding of this deadly virus and the associated infection.


Assuntos
Quirópteros , Doença do Vírus de Marburg , Marburgvirus , Animais , Modelos Animais de Doenças , Humanos , Doença do Vírus de Marburg/epidemiologia , Marburgvirus/fisiologia , Virulência
3.
Nat Commun ; 13(1): 1191, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246537

RESUMO

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex constitutes the core structure for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for helical assembly and subsequent viral RNA synthesis. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.


Assuntos
Ebolavirus , Marburgvirus , Microscopia Crioeletrônica , Ebolavirus/genética , Marburgvirus/genética , Nucleoproteínas/química , RNA Viral/química , RNA Viral/genética
4.
Antiviral Res ; 199: 105267, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35227759

RESUMO

The central role of Ebola virus (EBOV) VP40 in nascent virion assembly and budding from infected host cells makes it an important therapeutic target. The mechanism of dimerization, following oligomerization of VP40 leading to the production of virus-like particles (VLP) has never been investigated for the development of therapeutic candidates against Ebola disease. Molecular dynamics-based computational screening targeted VP40 dimer with 40,000,000 compounds selected 374 compounds. A novel in vitro screening assay selected two compounds, NUSU#1 and NUSU#2. Conventional VLP assays consistently showed that both compounds inhibited EBOV VP40-mediated VLP production. Intriguingly, NUSU#1 inhibited the VP40-mediated VLP production in other ebolavirus species and the Marburg virus, but did not inhibit Lassa virus Z-mediated VLP production. These results strongly suggested that the selected compounds are potential lead drug candidates against Filovirus disease via disruption of VP40-mediated particle production.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Marburgvirus , Ebolavirus/química , Humanos , Proteínas da Matriz Viral/química , Liberação de Vírus
5.
J Virol ; 96(6): e0202621, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35107375

RESUMO

Ebola virus (EBOV) and Marburg virus (MARV) continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW domain-containing host interactors via its conserved PPxY late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ, and WWOX. In addition to host WW domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 virus-like particles (VLPs) and recombinant vesicular stomatitis virus (VSV) M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOT's scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY-WW domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCE Filoviruses (Ebola virus [EBOV] and Marburg virus [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the filovirus matrix protein VP40 to regulate VP40-mediated egress of virus-like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus life cycle and the host cell.


Assuntos
Ebolavirus , Marburgvirus , Oxidorredutase com Domínios WW , /metabolismo , Ebolavirus/fisiologia , Humanos , Marburgvirus/metabolismo , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus/fisiologia , Oxidorredutase com Domínios WW/metabolismo
6.
Methods Mol Biol ; 2446: 427-449, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35157287

RESUMO

Fusions of single-domain antibodies (sdAbs, nanobodies) to enzymatic reporters make convenient molecular probes to detect the presence of an antigen of interest. We have previously fused the monomeric hyperactive ascorbate peroxidase derivative APEX2 to anti-Ebolavirus and anti-Marburgvirus sdAbs to generate immunoreagents useful in detecting nucleoprotein (NP) on western blots, ELISA, and within cells following transfection of NP expression plasmids or following virus infection. Here we present the methods used to overexpress and purify these sdAb-APEX2 fusion proteins, and to employ them as probes in various scenarios with colorimetric and fluorometric signal development. We also introduce a dimeric hyperactive ascorbate peroxidase derivative dEAPX that enables bivalent sdAb probes to be produced while avoiding the need to generate more complex tandem sdAbs, leveraging avidity for improved signal strength. The APEX2 and dEAPX reagents appear interchangeable with any existing detection platform and the methods described here should enable a user to study their antigen of interest by simply swapping out the sdAb for their recombinant affinity reagent of choice.


Assuntos
Ebolavirus , Marburgvirus , Anticorpos de Domínio Único , Ascorbato Peroxidases/genética , Nucleoproteínas , Anticorpos de Domínio Único/genética
8.
Expert Opin Drug Discov ; 17(2): 139-149, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34962451

RESUMO

INTRODUCTION: Filoviruses are negative-stranded, enveloped RNA viruses that can cause hemorrhagic fever in humans and include Ebola and Marburg viruses. Lethality rates can reach 90% in isolated outbreaks. The 2013-2016 Ebola virus epidemic demonstrated the global threat of filoviruses and hastened development of vaccines and therapeutics. There are six known filoviruses that cause disease in humans, but still few therapeutics are available for treatment. AREAS COVERED: This review summarizes identification, testing, and development of therapeutics based on the peer-reviewed scientific literature beginning with the discovery of filoviruses in 1967. Small molecules, antibodies, cytokines, antisense, post-exposure vaccination, and host-targeted therapeutic approaches are discussed. An emphasis is placed on therapeutics that have shown promise in in vivo studies. EXPERT OPINION: Two monoclonal antibody regimens are approved for use in humans for one filovirus (Ebola virus), and preclinical nonhuman primate studies suggest that other monoclonal-based therapies are likely to be effective against other filoviruses. Significant progress has been made in small-molecule antivirals and host-targeted approaches. An important consideration is the necessity of pan-filovirus therapeutics via broadly effective small molecules, antibody cocktails, and cross-reactive antibodies. The use of filovirus therapeutics as prophylactic treatment or in chronically infected individuals should be considered.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Marburgvirus , Animais , Anticorpos Monoclonais , Antivirais/farmacologia , Ebolavirus/genética , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/prevenção & controle
9.
Sci Rep ; 11(1): 24262, 2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34930962

RESUMO

Bat-borne viruses in the Henipavirus genus have been associated with zoonotic diseases of high morbidity and mortality in Asia and Australia. In Africa, the Egyptian rousette bat species (Rousettus aegyptiacus) is an important viral host in which Henipavirus-related viral sequences have previously been identified. We expanded these findings by assessing the viral dynamics in a southern African bat population. A longitudinal study of henipavirus diversity and excretion dynamics identified 18 putative viral species circulating in a local population, three with differing seasonal dynamics, and the winter and spring periods posing a higher risk of virus spillover and transmission. The annual peaks in virus excretion are most likely driven by subadults and may be linked to the waning of maternal immunity and recolonization of the roost in early spring. These results provide insightful information into the bat-host relationship that can be extrapolated to other populations across Africa and be communicated to at-risk communities as a part of evidence-based public health education and prevention measures against pathogen spillover threats.


Assuntos
Quirópteros/virologia , Reservatórios de Doenças/virologia , Marburgvirus/imunologia , Paramyxoviridae/imunologia , Estações do Ano , África , Animais , Ásia , Austrália , Geografia , Henipavirus , Humanos , Estudos Longitudinais , África do Sul , Fatores de Tempo , Zoonoses/epidemiologia , Zoonoses/virologia
10.
Viruses ; 13(12)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34960663

RESUMO

Marburg virus (MARV), the causative agent of Marburg virus disease, emerges sporadically in sub-Saharan Africa and is often fatal in humas. The natural reservoir for this zoonotic virus is the frugivorous Egyptian rousette bat (Rousettus aegyptiacus) that when infected, sheds virus in the highest amounts in oral secretions and urine. Being fruit bats, these animals forage nightly for ripened fruit throughout the year, including those types often preferred by humans. During feeding, they continually discard partially eaten fruit on the ground that could then be consumed by other Marburg virus susceptible animals or humans. In this study, using qRT-PCR and virus isolation, we tested fruit discarded by Egyptian rousette bats experimentally infected with a natural bat isolate of Marburg virus. We then separately tested viral persistence on fruit varieties commonly cultivated in sub-Saharan Africa using a recombinant Marburg virus expressing the fluorescent ZsGreen1. Marburg virus RNA was repeatedly detected on fruit in the food bowls of the infected bats and viable MARV was recovered from inoculated fruit for up to 6 h.


Assuntos
Quirópteros/virologia , Frutas/virologia , Marburgvirus/isolamento & purificação , Doenças dos Primatas/virologia , Zoonoses Virais/virologia , África ao Sul do Saara , Animais , Quirópteros/fisiologia , Quirópteros/urina , Reservatórios de Doenças/virologia , Humanos , Marburgvirus/classificação , Marburgvirus/genética , Doenças dos Primatas/transmissão , Primatas , Zoonoses Virais/transmissão
11.
PLoS Pathog ; 17(12): e1010151, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34914795

RESUMO

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.


Assuntos
Marburgvirus/enzimologia , Vírus da Parainfluenza 3 Humana/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Vírus Sinciciais Respiratórios/enzimologia , Proteínas do Complexo da Replicase Viral/metabolismo , Animais , Células Cultivadas
12.
Viruses ; 13(11)2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34835032

RESUMO

This study aimed to determine the vector competence of bat-associated nycteribiid flies (Eucamsipoda africana) for Marburg virus (MARV) in the Egyptian Rousette Bat (ERB), Rousettus aegyptiacus. In flies fed on subcutaneously infected ERBs and tested from 3 to 43 days post infection (dpi), MARV was detected only in those that took blood during the peak of viremia, 5-7 dpi. Seroconversion did not occur in control bats in contact with MARV-infected bats infested with bat flies up to 43 days post exposure. In flies inoculated intra-coelomically with MARV and tested on days 0-29 post inoculation, only those assayed on day 0 and day 7 after inoculation were positive by q-RT-PCR, but the virus concentration was consistent with that of the inoculum. Bats remained MARV-seronegative up to 38 days after infestation and exposure to inoculated flies. The first filial generation pupae and flies collected at different times during the experiments were all negative by q-RT-PCR. Of 1693 nycteribiid flies collected from a wild ERB colony in Mahune Cave, South Africa where the enzootic transmission of MARV occurs, only one (0.06%) tested positive for the presence of MARV RNA. Our findings seem to demonstrate that bat flies do not play a significant role in the transmission and enzootic maintenance of MARV. However, ERBs eat nycteribiid flies; thus, the mechanical transmission of the virus through the exposure of damaged mucous membranes and/or skin to flies engorged with contaminated blood cannot be ruled out.


Assuntos
Quirópteros/virologia , Dípteros/virologia , Vetores de Doenças , Marburgvirus/isolamento & purificação , Animais , Cavernas , Dípteros/fisiologia , Ectoparasitoses/veterinária , África do Sul
13.
Front Immunol ; 12: 774026, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34777392

RESUMO

Marburg virus (MARV) is a member of the filovirus family that causes hemorrhagic disease with high case fatality rates. MARV is on the priority list of the World Health Organization for countermeasure development highlighting its potential impact on global public health. We developed a vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) and previously demonstrated uniform protection of nonhuman primates (NHPs) with a single dose. Here, we investigated the fast-acting potential of this vaccine by challenging NHPs with MARV 14, 7 or 3 days after a single dose vaccination with VSV-MARV. We found that 100% of the animals survived when vaccinated 7 or 14 days and 75% of the animal survived when vaccinated 3 days prior to lethal MARV challenge. Transcriptional analysis of whole blood samples indicated activation of B cells and antiviral defense after VSV-MARV vaccination. In the day -14 and -7 groups, limited transcriptional changes after challenge were observed with the exception of day 9 post-challenge in the day -7 group where we detected gene expression profiles indicative of a recall response. In the day -3 group, transcriptional analysis of samples from surviving NHPs revealed strong innate immune activation. In contrast, the animal that succumbed to disease in this group lacked signatures of antiviral immunity. In summary, our data demonstrate that the VSV-MARV is a fast-acting vaccine suitable for the use in emergency situations like disease outbreaks in Africa.


Assuntos
Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Chlorocebus aethiops , Citocinas/sangue , Modelos Animais de Doenças , Imunização , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Ativação Linfocitária , Doença do Vírus de Marburg/sangue , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/metabolismo , Vacinação , Células Vero , Vesiculovirus , Carga Viral
14.
Front Immunol ; 12: 703986, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484200

RESUMO

Ebola (EBOV), Marburg (MARV) and Sudan (SUDV) viruses are the three filoviruses which have caused the most fatalities in humans. Transmission from animals into the human population typically causes outbreaks of limited scale in endemic regions. In contrast, the 2013-16 outbreak in several West African countries claimed more than 11,000 lives revealing the true epidemic potential of filoviruses. This is further emphasized by the difficulty seen with controlling the 2018-2020 outbreak of EBOV in the Democratic Republic of Congo (DRC), despite the availability of two emergency use-approved vaccines and several experimental therapeutics targeting EBOV. Moreover, there are currently no vaccine options to protect against the other epidemic filoviruses. Protection of a monovalent EBOV vaccine against other filoviruses has never been demonstrated in primate challenge studies substantiating a significant void in capability should a MARV or SUDV outbreak of similar magnitude occur. Herein we show progress on developing vaccines based on recombinant filovirus glycoproteins (GP) from EBOV, MARV and SUDV produced using the Drosophila S2 platform. The highly purified recombinant subunit vaccines formulated with CoVaccine HT™ adjuvant have not caused any safety concerns (no adverse reactions or clinical chemistry abnormalities) in preclinical testing. Candidate formulations elicit potent immune responses in mice, guinea pigs and non-human primates (NHPs) and consistently produce high antigen-specific IgG titers. Three doses of an EBOV candidate vaccine elicit full protection against lethal EBOV infection in the cynomolgus challenge model while one of four animals infected after only two doses showed delayed onset of Ebola Virus Disease (EVD) and eventually succumbed to infection while the other three animals survived challenge. The monovalent MARV or SUDV vaccine candidates completely protected cynomolgus macaques from infection with lethal doses of MARV or SUDV. It was further demonstrated that combinations of MARV or SUDV with the EBOV vaccine can be formulated yielding bivalent vaccines retaining full efficacy. The recombinant subunit vaccine platform should therefore allow the development of a safe and efficacious multivalent vaccine candidate for protection against Ebola, Marburg and Sudan Virus Disease.


Assuntos
Vacinas contra Ebola/farmacologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença do Vírus de Marburg/prevenção & controle , Marburgvirus/imunologia , Animais , Vacinas contra Ebola/genética , Vacinas contra Ebola/imunologia , Ebolavirus/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/imunologia , Humanos , Macaca fascicularis , Doença do Vírus de Marburg/epidemiologia , Doença do Vírus de Marburg/genética , Doença do Vírus de Marburg/imunologia , Marburgvirus/genética , Vacinas Sintéticas
15.
Antiviral Res ; 195: 105180, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34551346

RESUMO

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.


Assuntos
Adenina/análogos & derivados , Adenosina/análogos & derivados , Antivirais/farmacologia , Pirrolidinas/farmacologia , Adenina/farmacologia , Adenosina/farmacologia , Animais , Ensaios Clínicos Fase I como Assunto , Avaliação Pré-Clínica de Medicamentos , Marburgvirus/efeitos dos fármacos , Nucleosídeos/análogos & derivados , SARS-CoV-2/efeitos dos fármacos
16.
J Virol ; 95(19): e0065221, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34346762

RESUMO

The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3' UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5' or 3' UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3' UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3' UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3' UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3' UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3' UTR, but not the edited or deletion mutant NP 3' UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3' UTR, VP40 3' UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3' UTR edits also enhanced translation, but neither the wild-type nor the edited 3' UTRs induced IFN. These findings implicate filoviral mRNA 3' UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3' UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3' UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3' UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.


Assuntos
Regiões 3' não Traduzidas , Adenosina Desaminase/metabolismo , Ebolavirus/genética , Marburgvirus/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Ebolavirus/metabolismo , Genes Reporter , Humanos , Interferon Tipo I/biossíntese , Marburgvirus/metabolismo , MicroRNAs/genética , Mutação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Polirribossomos/metabolismo , Edição de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
17.
Viruses ; 13(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209295

RESUMO

Ebolaviruses and marburgviruses are filoviruses that are known to cause severe hemorrhagic fever in humans and nonhuman primates (NHPs). While some bat species are suspected to be natural reservoirs of these filoviruses, wild NHPs often act as intermediate hosts for viral transmission to humans. Using an enzyme-linked immunosorbent assay, we screened two NHP species, wild baboons and vervet monkeys captured in Zambia, for their serum IgG antibodies specific to the envelope glycoproteins of filoviruses. From 243 samples tested, 39 NHPs (16%) were found to be seropositive either for ebolaviruses or marburgviruses with endpoint antibody titers ranging from 100 to 25,600. Interestingly, antibodies reactive to Reston virus, which is found only in Asia, were detected in both NHP species. There was a significant difference in the seropositivity for the marburgvirus antigen between the two NHP species, with baboons having a higher positive rate. These results suggest that wild NHPs in Zambia might be nonlethally exposed to these filoviruses, and this emphasizes the need for continuous monitoring of filovirus infection in wild animals to better understand the ecology of filoviruses and to assess potential risks of outbreaks in humans in previously nonendemic countries.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Filoviridae/imunologia , Infecções por Filoviridae/veterinária , Filoviridae/imunologia , Primatas/virologia , Animais , Animais Selvagens/virologia , Chlorocebus aethiops/virologia , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Filoviridae/classificação , Filoviridae/isolamento & purificação , Infecções por Filoviridae/epidemiologia , Humanos , Imunoglobulina G/sangue , Masculino , Marburgvirus/imunologia , Papio/virologia , Estudos Soroepidemiológicos , Zâmbia/epidemiologia
18.
J Clin Lab Anal ; 35(6): e23786, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33939238

RESUMO

BACKGROUND: Marburg virus (MARV) and Ebola virus (EBOV) are acute infections with high case fatality rates. It is of great significance for epidemic monitoring and prevention and control of infectious diseases by the development of a rapid, specific, and sensitive quantitative PCR method to detect two pathogens simultaneously. METHODS: Primers and TaqMan probes were designed according to highly conserved sequences of these viruses. Sensitivity, specificity, linear range, limit of detection, and the effects of hemolysis and lipid on real-time qPCR were evaluated. RESULTS: The linearity of the curve allowed quantification of nucleic acid concentrations in range from 103 to 109  copies/ml per reaction (MARV and EBOV). The limit of detection of EBOV was 40 copies/ml, and MARV was 100 copies/ml. It has no cross-reaction with other pathogens such as hepatitis b virus (HBV), hepatitis c virus (HCV), human papillomavirus (HPV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Repeatability analysis of the two viruses showed that their coefficient of variation (CV) was less than 5.0%. The above results indicated that fluorescence quantitative PCR could detect EBOV and MARV sensitively and specifically. CONCLUSIONS: The TaqMan probe-based multiplex fluorescence quantitative PCR assays could detect EBOV and MARV sensitively specifically and simultaneously.


Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/diagnóstico , Doença do Vírus de Marburg/diagnóstico , Marburgvirus/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Doença pelo Vírus Ebola/virologia , Humanos , Doença do Vírus de Marburg/virologia , Curva ROC
19.
J Biol Chem ; 296: 100796, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019871

RESUMO

Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein-protein or lipid-protein interactions to inhibit virus budding.


Assuntos
Doença do Vírus de Marburg/virologia , Marburgvirus/fisiologia , Lipídeos de Membrana/metabolismo , Proteínas da Matriz Viral/metabolismo , Vírion/metabolismo , Animais , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Doença do Vírus de Marburg/metabolismo , Marburgvirus/química , Lipídeos de Membrana/química , Modelos Moleculares , Multimerização Proteica , Proteínas da Matriz Viral/química , Vírion/química , Montagem de Vírus
20.
Antimicrob Agents Chemother ; 65(7): e0008621, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33846137

RESUMO

Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW domain-containing proteins via its conserved PPxY late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small-molecule compounds targeting the viral PPxY/host WW domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure-activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of mVP40 virus-like particles (VLPs) and egress of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated-mice displayed delayed onset of weight loss and clinical signs and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV life cycle.


Assuntos
Marburgvirus , Animais , Células HeLa , Humanos , Camundongos , Liberação de Vírus
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