RESUMEN
Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5' and 3' ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5' end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5' end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.
Asunto(s)
Ebolavirus , Filoviridae , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Viral , Análisis de Secuencia de ARN , ARN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ebolavirus/genética , Análisis de Secuencia de ARN/métodos , Filoviridae/genética , Marburgvirus/genética , Humanos , AnimalesRESUMEN
Ebola virus (EBOV) and Marburg virus (MARV), members of the Filoviridae family, are highly pathogenic and can cause hemorrhagic fevers, significantly impacting human society. Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats. However, due to the requirement for maximum containment laboratories when studying infectious viruses, the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems. In this study, we used RACE technology to sequence the 3'-leader and 5'-trailer of Menglà virus (MLAV) and constructed a minigenome. Similar to MARV, the transcription activities of the MLAV minigenome are independent of VP30. We further assessed the effects of polymorphisms at the 5' end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency, probably due to alterations in the RNA secondary structure. The reporter activity upon recombination of the 3'-leaders and 5'-trailers of MLAV, MARV, and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities. Additionally, we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system. Remdesivir efficiently inhibited MLAV as well as EBOV replication. In summary, this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.
Asunto(s)
Ebolavirus , Genoma Viral , Marburgvirus , Animales , Genoma Viral/genética , Ebolavirus/genética , Humanos , Marburgvirus/genética , Mengovirus/genética , Replicación Viral , ARN Viral/genética , Alanina/análogos & derivados , Alanina/farmacología , Quirópteros/virología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/metabolismo , Filoviridae/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Paramyxo- and filovirus genomes are equipped with bipartite promoters at their 3' ends to initiate RNA synthesis. The two elements, the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2), are separated by a spacer region that must be precisely a multiple of 6 nucleotides (nts), indicating these viruses adhere to the "rule of six." However, our knowledge of PE2 has been limited to a narrow spectrum of virus species. In this study, a comparative analysis of 1,647 paramyxoviral genomes from a public database revealed that the paramyxovirus PE2 can be clearly categorized into two distinct subcategories: one marked by C repeats at every six bases (exclusive to the subfamily Orthoparamyxovirinae) and another characterized by CG repeats every 6 nts (observed in the subfamilies Avulavirinae and Rubulavirinae). This unique pattern collectively mirrors the evolutionary lineage of these subfamilies. Furthermore, we showed that PE2 of the Rubulavirinae, with the exception of mumps virus, serves as part of the gene-coding region. This may be due to the fact that the Rubulavirinae are the only paramyxoviruses that cannot propagate without RNA editing. Filoviruses have three to eight consecutive uracil repeats every six bases (UN5) in PE2, which is located in the 3' end region of the genome. We obtained PE2 sequences from 2,195 filoviruses in a public database and analyzed the sequence conservation among virus species. Our results indicate that the continuity of UN5 hexamers is consistently maintained with a high degree of conservation across virus species. IMPORTANCE: The genomic intricacies of paramyxo- and filoviruses are highlighted by the bipartite promoters-promoter element 1 (PE1) and promoter element 2 (PE2)-at their 3' termini. The spacer region between these elements follows the "rule of six," crucial for genome replication. By a comprehensive analysis of paramyxoviral genome sequences, we identified distinct subcategories of PE2 based on C and CG repeats that were specific to Orthoparamyxovirinae and Avulavirinae/Rubulavirinae, respectively, mirroring their evolutionary lineages. Notably, the PE2 of Rubulavirinae is integrated into the gene-coding region, a unique trait potentially linked to its strict dependence on RNA editing for virus growth. This study also focused on the PE2 sequences in filovirus genomes. The strict conservation of the continuity of UN5 among virus species emphasizes its crucial role in viral genome replication.
Asunto(s)
Filoviridae , Genoma Viral , Filogenia , Regiones Promotoras Genéticas , Regiones Promotoras Genéticas/genética , Genoma Viral/genética , Filoviridae/genética , Filoviridae/clasificación , Paramyxoviridae/genética , Paramyxoviridae/clasificación , Humanos , ARN Viral/genética , Evolución Molecular , AnimalesRESUMEN
Filoviruses are causative agents of severe hemorrhagic fevers with high case fatality rates in humans. For studies of virus biology and the subsequent development of countermeasures, reverse genetic systems, and especially those facilitating the generation of recombinant filoviruses, are indispensable. Here, we describe the generation of recombinant filoviruses from cDNA.
Asunto(s)
Ebolavirus , Filoviridae , Fiebre Hemorrágica Ebola , Humanos , Filoviridae/genética , Genética Inversa , ADN Complementario/genética , Ebolavirus/genéticaRESUMEN
Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses.
Asunto(s)
Quirópteros , Filoviridae , Marburgvirus , Animales , Filoviridae/genética , Línea Celular , Italia , FilogeniaRESUMEN
Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no pan-filovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3-31.7 copies/reaction and was evaluated against the field collected samples. The assay's performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples.
Asunto(s)
Biovigilancia , Quirópteros , Ebolavirus , Filoviridae , Fiebre Hemorrágica Ebola , Animales , Filoviridae/genética , Ebolavirus/genética , MamíferosRESUMEN
Several filoviruses, including Marburg virus (MARV), cause severe disease in humans and nonhuman primates (NHPs). However, the Egyptian rousette bat (ERB, Rousettus aegyptiacus), the only known MARV reservoir, shows no overt illness upon natural or experimental infection, which, like other bat hosts of zoonoses, is due to well-adapted, likely species-specific immune features. Despite advances in understanding reservoir immune responses to filoviruses, ERB peripheral blood responses to MARV and how they compare to those of diseased filovirus-infected spillover hosts remain ill-defined. We thus conducted a longitudinal analysis of ERB blood gene responses during acute MARV infection. These data were then contrasted with a compilation of published primate blood response studies to elucidate gene correlates of filovirus protection versus disease. Our work expands on previous findings in MARV-infected ERBs by supporting both host resistance and disease tolerance mechanisms, offers insight into the peripheral immunocellular repertoire during infection, and provides the most direct known cross-examination between reservoir and spillover hosts of the most prevalently-regulated response genes, pathways and activities associated with differences in filovirus pathogenesis and pathogenicity.
Asunto(s)
Quirópteros , Filoviridae , Marburgvirus , Humanos , Animales , Filoviridae/genética , Tolerancia Inmunológica , InmunidadRESUMEN
Emerging infectious diseases, especially if caused by bat-borne viruses, significantly affect public health and the global economy. There is an urgent need to understand the mechanism of interspecies transmission, particularly to humans. Viral genetics; host factors, including polymorphisms in the receptors; and ecological, environmental, and population dynamics are major parameters to consider. Here, we describe the taxonomy, geographic distribution, and unique traits of bats associated with their importance as virus reservoirs. Then, we summarize the origin, intermediate hosts, and the current understanding of interspecies transmission of Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, Nipah, Hendra, Ebola, Marburg virus, and rotaviruses. Finally, the molecular interactions of viral surface proteins with host cell receptors are examined, and a comparison of these interactions in humans, intermediate hosts, and bats is conducted. This uncovers adaptive mutations in virus spike protein that facilitate cross-species transmission and risk factors associated with the emergence of novel viruses from bats.
Asunto(s)
COVID-19 , Quirópteros , Filoviridae , Henipavirus , Rotavirus , Virus , Animales , Filoviridae/genética , Humanos , Rotavirus/genética , SARS-CoV-2/genéticaRESUMEN
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.
Asunto(s)
Ebolavirus/genética , Infecciones por Filoviridae/virología , Filoviridae/genética , Replicación Viral , Animales , Línea Celular , Chlorocebus aethiops , Prueba de Complementación Genética , Genoma Viral , Fiebre Hemorrágica Ebola/virología , Interacciones Microbiota-Huesped , Humanos , Cuerpos de Inclusión/virología , Células Madre Pluripotentes Inducidas/virología , Macrófagos/virología , ARN Viral , Genética Inversa , Células Vero , Virión/genéticaRESUMEN
I performed metaviromic analysis of publicly available RNA-seq data from reptiles to understand the diversity of filoviruses (family Filoviridae). I identified a coding-complete sequence of a filovirus from the common lancehead (Bothrops atrox (Linnaeus, 1758)), tentatively named Tapajós virus (TAPV). Although the genome organization of TAPV is similar to mammalian filoviruses, our phylogenetic analysis showed that TAPV forms a cluster with a fish filovirus. However, TAPV is still distantly related to all the known filoviruses, suggesting that TAPV can be assigned as a species of a novel genus in Filoviridae. To our knowledge, this is the first report identifying a filovirus in reptiles, and thus contributes to a deeper understanding of the diversity and evolution of filoviruses.
Asunto(s)
Bothrops , Filoviridae , Animales , Filoviridae/genética , Genoma , Mamíferos , FilogeniaRESUMEN
In recent years, a number of novel filoviruses (e.g. Lloviu virus (LLOV) and Bombali virus (BOMV)) have been discovered. While antibody-based therapeutics have recently been approved for treatment of infections with the filovirus Ebola virus (EBOV), no treatment options for novel filoviruses currently exist. Further, the development of antivirals against them is complicated by the fact that only sequence information, but no actual virus isolates, are available. To address this issue, we developed a reverse genetics-based minigenome system for BOMV, which allows us to assess the activity of the BOMV polymerase. Together with similar systems that we have developed for other filoviruses in the past (i.e. LLOV and Reston virus (RESTV)), we then assessed the efficiency of remdesivir, a known inhibitor of the EBOV polymerase that has recently been tested in a clinical trial for efficacy against Ebola disease. We show that remdesivir is indeed also active against the polymerases of BOMV, LLOV, and RESTV, with comparable IC50 values to its activity against EBOV. This suggests that treatment with remdesivir might represent a viable option in case of infections with novel filoviruses.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/farmacología , Filoviridae/efectos de los fármacos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Adenosina Monofosfato/farmacología , Alanina/farmacología , Línea Celular , Ebolavirus/efectos de los fármacos , Filoviridae/clasificación , Filoviridae/genética , Humanos , Concentración 50 Inhibidora , Filogenia , Replicación Viral/efectos de los fármacosRESUMEN
Filovirus family consists of highly pathogenic viruses that have caused fatal outbreaks especially in many African countries. Previously, research focus has been on Ebola, Sudan and Marburg viruses leaving other filoviruses less well studied. Filoviruses, in general, pose a significant global threat since they are highly virulent and potentially transmissible between humans causing sporadic infections and local or widespread epidemics. Filoviruses have the ability to downregulate innate immunity, and especially viral protein 24 (VP24), VP35 and VP40 have variably been shown to interfere with interferon (IFN) gene expression and signaling. Here we systematically analyzed the ability of VP24 proteins of nine filovirus family members to interfere with retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated antigen 5 (MDA5) induced IFN-ß and IFN-λ1 promoter activation. All VP24 proteins were localized both in the cell cytoplasm and nucleus in variable amounts. VP24 proteins of Zaire and Sudan ebolaviruses, Lloviu, Taï Forest, Reston, Marburg and Bundibugyo viruses (EBOV, SUDV, LLOV, TAFV, RESTV, MARV and BDBV, respectively) were found to inhibit both RIG-I and MDA5 stimulated IFN-ß and IFN-λ1 promoter activation. The inhibition takes place downstream of interferon regulatory factor 3 phosphorylation suggesting the inhibition to occur in the nucleus. VP24 proteins of Mengla (MLAV) or Bombali viruses (BOMV) did not inhibit IFN-ß or IFN-λ1 promoter activation. Six ebolavirus VP24s and Lloviu VP24 bound tightly, whereas MARV and MLAV VP24s bound weakly, to importin α5, the subtype that regulates the nuclear import of STAT complexes. MARV and MLAV VP24 binding to importin α5 was very weak. Our data provides new information on the innate immune inhibitory mechanisms of filovirus VP24 proteins, which may contribute to the pathogenesis of filovirus infections.
Asunto(s)
Proteína 58 DEAD Box/inmunología , Filoviridae/inmunología , Interferón Tipo I/inmunología , Helicasa Inducida por Interferón IFIH1/inmunología , Interferones/inmunología , Interleucinas/inmunología , Regiones Promotoras Genéticas/inmunología , Receptores Inmunológicos/inmunología , Proteínas Virales/inmunología , Línea Celular Tumoral , Proteína 58 DEAD Box/genética , Filoviridae/genética , Regulación de la Expresión Génica/inmunología , Células HEK293 , Humanos , Interferón Tipo I/genética , Helicasa Inducida por Interferón IFIH1/genética , Interferones/genética , Interleucinas/genética , Receptores Inmunológicos/genética , Proteínas Virales/genéticaRESUMEN
The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.
Asunto(s)
Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Fosfatidilinositol 3-Quinasa/genética , Proteínas Proto-Oncogénicas c-akt/genética , Infecciones por Virus ARN/genética , Apoptosis/genética , Autofagia/genética , Autofagia/inmunología , Citocinas/genética , Citocinas/inmunología , Endocitosis/genética , Endocitosis/inmunología , Filoviridae/genética , Filoviridae/metabolismo , Filoviridae/patogenicidad , Interacciones Huésped-Patógeno/inmunología , Interferones/genética , Interferones/inmunología , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Orthomyxoviridae/patogenicidad , Paramyxoviridae/genética , Paramyxoviridae/metabolismo , Paramyxoviridae/patogenicidad , Fosfatidilinositol 3-Quinasa/inmunología , Pneumovirinae/genética , Pneumovirinae/metabolismo , Pneumovirinae/patogenicidad , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-akt/inmunología , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Rhabdoviridae/genética , Rhabdoviridae/metabolismo , Rhabdoviridae/patogenicidad , Transducción de Señal , Proteínas Virales/genética , Proteínas Virales/inmunología , Internalización del Virus , Replicación ViralRESUMEN
Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH-activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP), exposing a domain which binds to the filoviral receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance-associated gene (UVRAG). Using an inducible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression of and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry.IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly ones with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest, as all filoviruses must reach cellular compartments containing the viral receptor Niemann-Pick C1 to enter cells. Here, we present an inducible CRISPR/Cas9 method to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering complex in coordination with UV radiation resistance-associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.
Asunto(s)
Ebolavirus/metabolismo , Proteína Niemann-Pick C1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Ebolavirus/genética , Ebolavirus/patogenicidad , Endosomas/metabolismo , Filoviridae/genética , Infecciones por Filoviridae/genética , Infecciones por Filoviridae/metabolismo , Glicoproteínas/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Interacciones Huésped-Patógeno , Glicoproteínas de Membrana/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Receptores Virales/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas del Envoltorio Viral/genética , Internalización del Virus/efectos de los fármacosRESUMEN
Menglà virus (MLAV), identified in Rousettus bats, is a phylogenetically distinct member of the family Filoviridae Because the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) modulate host innate immunity, MLAV VP35, VP40, and VP24 proteins were compared with their EBOV and MARV homologs for innate immune pathway modulation. In human and Rousettus cells, MLAV VP35 behaved like EBOV and MARV VP35s, inhibiting virus-induced activation of the interferon beta (IFN-ß) promoter and interferon regulatory factor 3 (IRF3) phosphorylation. MLAV VP35 also interacted with PACT, a host protein engaged by EBOV VP35 to inhibit RIG-I signaling. MLAV VP35 also inhibits PKR activation. MLAV VP40 was demonstrated to inhibit type I IFN-induced gene expression in human and bat cells. It blocked STAT1 tyrosine phosphorylation induced either by type I IFN or overexpressed Jak1, paralleling MARV VP40. MLAV VP40 also inhibited virus-induced IFN-ß promoter activation, a property shared by MARV VP40 and EBOV VP24. A Jak kinase inhibitor did not recapitulate this inhibition in the absence of viral proteins. Therefore, inhibition of Jak-STAT signaling is insufficient to explain inhibition of IFN-ß promoter activation. MLAV VP24 did not inhibit IFN-induced gene expression or bind karyopherin α proteins, properties of EBOV VP24. MLAV VP24 differed from MARV VP24 in that it failed to interact with Keap1 or activate an antioxidant response element reporter gene due to the absence of a Keap1-binding motif. These functional observations support a closer relationship of MLAV to MARV than to EBOV but also are consistent with MLAV belonging to a distinct genus.IMPORTANCE EBOV and MARV, members of the family Filoviridae, are highly pathogenic zoonotic viruses that cause severe disease in humans. Both viruses use several mechanisms to modulate the host innate immune response, and these likely contribute to the severity of disease. Here, we demonstrate that MLAV, a filovirus newly discovered in a bat, suppresses antiviral type I interferon responses in both human and bat cells. Inhibitory activities are possessed by MLAV VP35 and VP40, which parallels how MARV blocks IFN responses. However, whereas MARV activates cellular antioxidant responses through an interaction between its VP24 protein and host protein Keap1, MLAV VP24 lacks a Keap1-binding motif and fails to activate this cytoprotective response. These data indicate that MLAV possesses immune-suppressing functions that could facilitate human infection. They also support the placement of MLAV in a different genus than either EBOV or MARV.
Asunto(s)
Infecciones por Filoviridae/fisiopatología , Filoviridae/genética , Animales , Quirópteros/inmunología , Quirópteros/virología , Ebolavirus , Filoviridae/metabolismo , Filoviridae/patogenicidad , Células HEK293 , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/inmunología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Marburgvirus , Factor 2 Relacionado con NF-E2/metabolismo , Factor de Transcripción STAT1 , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
A serological survey of 2,430 archived serum samples collected between 1997 and 2012 was conducted to retrospectively determine the prevalence of Marburg virus in five African countries. Serum samples were screened for neutralizing antibodies in a pseudotype micro-neutralization assay and confirmed by enzyme-linked immunosorbent assay (ELISA). Surprisingly, a seroprevalence for Marburg virus of 7.5 and 6.3% was found in Cameroon and Ghana, respectively, suggesting the circulation of filoviruses or related viruses outside of known endemic areas that remain undetected by current surveillance efforts. However, due to the lack of validated assays and appropriate positive controls, these results must be considered preliminary.
Asunto(s)
Anticuerpos Antivirales/sangre , Filoviridae/inmunología , Enfermedad del Virus de Marburg/sangre , Enfermedad del Virus de Marburg/epidemiología , Marburgvirus/inmunología , Animales , Camerún/epidemiología , Ensayo de Inmunoadsorción Enzimática , Filoviridae/genética , Infecciones por Filoviridae/sangre , Infecciones por Filoviridae/epidemiología , Infecciones por Filoviridae/virología , Ghana/epidemiología , Humanos , Enfermedad del Virus de Marburg/virología , Marburgvirus/genética , Estudios Retrospectivos , Estudios SeroepidemiológicosRESUMEN
Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.
Asunto(s)
Filoviridae/genética , Proteína Niemann-Pick C1/metabolismo , Tropismo/genética , Secuencia de Aminoácidos , Animales , Quirópteros , Humanos , Modelos MolecularesRESUMEN
Viral hemorrhagic fevers (VHFs) cause thousands of fatalities every year, but the treatment options for their management remain very limited. In particular, the development of therapeutic interventions is restricted by the lack of commercial viability of drugs targeting individual VHF agents. This makes approaches like drug repurposing and/or the identification of broad range therapies (i.e. those directed at host responses or common proviral factors) highly attractive. However, the identification of candidates for such antiviral repurposing or of host factors/pathways important for the virus life cycle is reliant on high-throughput screening (HTS). Recently, such screening work has been increasingly facilitated by the availability of reverse genetics-based approaches, including tools such as full-length clone (FLC) systems to generate reporter-expressing viruses or various life cycle modelling (LCM) systems, many of which have been developed and/or greatly improved during the last years. In particular, since LCM systems are capable of modelling specific steps in the life cycle, they are a valuable tool for both targeted screening (i.e. for inhibitors of a specific pathway) and mechanism of action studies. This review seeks to summarize the currently available reverse genetics systems for negative-sense VHF causing viruses (i.e. arenaviruses, bunyaviruses and filoviruses), and to highlight the recent advancements made in applying these systems for HTS to identify either antivirals or new virus-host interactions that might hold promise for the development of future treatments for the infections caused by these deadly but neglected virus groups.
Asunto(s)
Arenaviridae/genética , Bunyaviridae/genética , Filoviridae/genética , Fiebres Hemorrágicas Virales/virología , Ensayos Analíticos de Alto Rendimiento , Genética Inversa/métodos , Antivirales/aislamiento & purificación , Arenaviridae/efectos de los fármacos , Bunyaviridae/efectos de los fármacos , Filoviridae/efectos de los fármacos , Genoma Viral , Interacciones Microbiota-Huesped , HumanosRESUMEN
Research on filoviruses has historically focused on the highly pathogenic ebola- and marburgviruses. Indeed, until recently, these were the only two genera in the filovirus family. Recent advances in sequencing technologies have facilitated the discovery of not only a new ebolavirus, but also three new filovirus genera and a sixth proposed genus. While two of these new genera are similar to the ebola- and marburgviruses, the other two, discovered in saltwater fishes, are considerably more diverse. Nonetheless, these viruses retain a number of key features of the other filoviruses. Here, we review the key characteristics of filovirus replication and transcription, highlighting similarities and differences between the viruses. In particular, we focus on key regulatory elements in the genomes, replication and transcription strategies, and the conservation of protein domains and functions among the viruses. In addition, using computational analyses, we were able to identify potential homology and functions for some of the genes of the novel filoviruses with previously unknown functions. Although none of the newly discovered filoviruses have yet been isolated, initial studies of some of these viruses using minigenome systems have yielded insights into their mechanisms of replication and transcription. In general, the Cuevavirus and proposed Dianlovirus genera appear to follow the transcription and replication strategies employed by the ebola- and marburgviruses, respectively. While our knowledge of the fish filoviruses is currently limited to sequence analysis, the lack of certain conserved motifs and even entire genes necessitates that they have evolved distinct mechanisms of replication and transcription.
Asunto(s)
Filoviridae/genética , Genoma Viral/genética , Elementos Reguladores de la Transcripción/genética , Elementos Reguladores de la Transcripción/fisiología , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein-protein interactions on a functional level.