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Three ebolaviruses (Bundibugyo virus [BDBV], Ebola virus [EBOV], and Sudan virus [SUDV]) are highly lethal human pathogens. Taï Forest virus (TAFV) has caused a single case of severe but nonlethal human disease, whereas Reston virus (RESTV) has thus far only caused inapparent human infections (Kuhn 2018). Ebolaviruses are notable for encoding three distinct proteins from their glycoprotein (GP) genes, a strategy they share with cuevaviruses (Negredo et al., 2011, Sanchez et al., 1996, Volchkov et al., 1995).
Virions are filamentous in shape, but can also be branched, circular, U- or 6-shaped. Spherical forms are rare to absent (Figure 1.Ebolavirus). Virions vary greatly in length (>20 μm) but have a diameter of 96-98 nm. Peak infectivity has been associated with particles of ≈805 nm in length (Beniac et al., 2012, Ellis et al., 1979a, Ellis et al., 1979b, Geisbert and Jahrling 1995, Ryabchikova and Price 2004).
Virions are composed of a central core formed by a helical ribonucleoprotein (RNP) complex, surrounded by matrix layer and a lipid envelope derived from the host-cell plasma membrane. Polyploidy has been observed. Spikes ≈7 nm in diameter and spaced at intervals of ≈10 nm are seen as globular structures on the surface of virions (Beniac et al., 2012, Geisbert and Jahrling 1995, Ryabchikova and Price 2004, Sugita et al., 2018).
Figure 1.Ebolavirus. A) Scanning electron microscograph of Ebola virus particles (blue) budding from an infected grivet (Chlorocebus aethiops Linnaeus, 1758) Vero E6 cell. B) Transmission electron microscograph of Ebola virus particles (blue) found both as extracellular particles and budding particles from infected Vero E6 cells. Images are colorized for clarity. Courtesy of John G. Bernbaum and Jiro Wada, IRF-Frederick, Fort Detrick, MD, USA.
EBOV particles have a molecular mass of ≈3.82×105 kD. The buoyant density of EBOV particles in potassium tartrate is ≈1.14 g/ml. The sedimentation coefficient (S20,w) of uniform filamentous particles is 1,300–1,400 S, but higher for longer particles. The nucleocapsid has a buoyant density in CsCl of ≈1.32 g cm−3 (Kiley et al., 1980).
Ebolavirus genomes are non-segmented, linear RNA molecules of negative polarity. The genomes are ≈18.9 kb. Genomic RNAs are not polyadenylated at their 3′-ends and there is no evidence for 5′-terminal cap structures or covalently-linked proteins. The Mr of genomic RNA is about 4.2×106 and represents about 1.1% of the total virion mass (Kiley et al., 1980, Regnery et al., 1980, Elliott et al., 1985, Boehmann et al., 2005).
Ebolaviruses express 7 structural proteins, all of which are homologous to those of cuevaviruses and marburgviruses. The second most abundant structural protein in virions is the nucleoprotein (NP), which encapsidates the ebolavirus genome. The least abundant protein is the RNA-dependent RNA polymerase (L), which mediates ebolavirus genome replication and transcription (Sanchez et al., 1993, Kiley et al., 1980). The ebolavirus RNP complex consists of NP, RNP complex-associated protein (VP24), polymerase cofactor (VP35), transcriptional activator (VP30), and L. The RNP complexes associate with the matrix proteins (VP40), which line the inner side of the virion membrane, and glycoproteins (GP1,2), which form globular spikes on the outside of the virion membrane (Beniac et al., 2012, Sugita et al., 2018, Kirchdoerfer et al., 2017, Mühlberger et al., 1999, Banadyga et al., 2017). Similar to cuevaviruses, but unlike marburgviruses, ebolaviruses express their structural glycoproteins (GP1,2) via co-transcriptional editing and also express three soluble glycoproteins from the GP gene (sGP, ssGP, and Δ-peptide) (Sanchez et al., 1996, Volchkov et al., 1995, Volchkova et al., 1999, Mehedi et al., 2011).
Table 1.Ebolavirus. Location and functions of ebolavirus structural proteins.
RNP complex component; second-most abundant protein in infected cells and in virons; consists of two distinct functional modules; homo-oligomerizes to form helical polymers; binds to genomic and antigenomic RNA, VP35, VP40, VP30, and VP24; phosphorylated, O-glycosylated, and possibly sialylated
Nucleocapsid and cellular inclusion body formation; encapsidation of filovirus genome and antigenome; genome replication and transcription
(Sugita et al., 2018, Mühlberger et al., 1999, Su et al., 2018, Watanabe et al., 2006, Peyrol et al., 2013, Groseth et al., 2009, Hoenen et al., 2012, Ebihara et al., 2006, Huang et al., 2002, Kirchdoerfer et al., 2015)
Polymerase cofactor (VP35)
RNP complex component; homo-oligomer; phosphorylated; binds to double-stranded RNA, NP, and L
Replicase-transcriptase cofactor; inhibits innate immune response by interfering with IRF-3 and IRF-7, and MDA-5, RIG-1, and RNAi pathways; inhibits stress granule formation
(Mühlberger et al., 1999, Kirchdoerfer et al., 2015, Basler et al., 2003, Basler et al., 2000, Cárdenas et al., 2006, Haasnoot et al., 2007, Kimberlin et al., 2010, Leung et al., 2010, Reid et al., 2005, Nelson et al., 2016)
Matrix protein (VP40)
Most abundant protein in infected cells and in virons; consists of two distinct functional modules; homo-oligomerizes to form dimers and circular hexamers and octamers; SUMOylated; binds single-stranded RNA, α-tubulin, VP35; hydrophobic; membrane-associated; contains one (marburgviruses) or three (cuevaviruses and ebolaviruses) late-budding motifs; binds NEDD4 and Tsg101; ubiquitinylated
Matrix component; regulation of genome transcription and replication; regulation of virion morphogenesis and egress
(Bornholdt et al., 2013, Harty et al., 2000, Licata et al., 2003, Noda et al., 2002, Baz-Martínez et al., 2016, Clifton et al., 2015, Dessen et al., 2000, Hoenen et al., 2010, Panchal et al., 2003, Pavadai et al., 2018, Timmins et al., 2003)
Secreted glycoprotein (sGP)
Mostly nonstructural; secreted as a parallel homo-dimer in high amounts from infected cells; N-glycosylated, C-mannosylated
Unknown. Hypothesized to be an antibody-decoy and an anti-inflammatory agent
(Falzarano et al., 2007, Volchkova et al., 1998, Barrientos et al., 2004, Falzarano et al., 2006, de La Vega et al., 2015)
Spike glycoprotein (GP1,2)
Type I transmembrane and class I fusion protein; cleaved to GP1 and GP2 subunits that heterodimerize; mature protein is a trimer of GP1,2 heterodimers; inserts into membranes; heavily N- and O-glycosylated, sialylated, acylated, phosphorylated. Tumor necrosis factor α-converting enzyme (TACE) converts GP1,2 into a soluble form (GP1,2Δ)
Virion adsorption to filovirus-susceptible cells via cellular attachment factors; determines filovirus cell and tissue tropism; induction of virus-cell membrane fusion subsequent to endolysosomal binding to NPC1; inhibits innate immune response by interfering with tetherin. Function of GP1,2Δ is unknown
(Ito et al., 2001, Carette et al., 2011, Côté et al., 2011, Chandran et al., 2005, Schornberg et al., 2006, Lee et al., 2008, Collar et al., 2017, Ritchie et al., 2010, Beniac and Booth 2017, Jeffers et al., 2002, Kaletsky et al., 2009, Malashkevich et al., 1999, Tran et al., 2014, Volchkov et al., 1998a, Volchkov et al., 1998b, Wang et al., 2016, Jouvenet et al., 2009, Dolnik et al., 2004, Takada et al., 1997)
Secondary secreted glycoprotein (ssGP)
Predominantly nonstructural; secreted as a N-glycosylated monomer
(Mehedi et al., 2011, Volchkova et al., 1998)
Nonstructural; secreted; largely unstructured; O-glycosylated and sialylated
Hypothesized to suppress filovirus superinfection and/or act as a viroporin
(Radoshitzky et al., 2011, Volchkova et al., 1999, He et al., 2017)
Transcriptional activator (VP30)
RNP complex component; hexameric zinc finger protein; binds single-stranded RNA, NP, and L; phosphorylated
Transcription initiation, reinitiaition, and antitermination
(Mühlberger et al., 1999, Groseth et al., 2009, Biedenkopf et al., 2013, Biedenkopf et al., 2016a, Biedenkopf et al., 2016b, John et al., 2007, Modrof et al., 2003, Modrof et al., 2002, Xu et al., 2017, Weik et al., 2002)
RNP complex-associated protein (VP24)
RNP complex component; homo-tetramerizes; hydrophobic and membrane-associated
Regulation of genome transcription and replication; regulation of virion morphogenesis and egress; inhibits phosphorylation of MAPK and prevents karyopherin shuttling from cytoplasm into the nucleus; inhibis host-cell signaling downstream of IFN-α/β/γ
(Banadyga et al., 2017, Ebihara et al., 2006, Halfmann et al., 2011, Han et al., 2003, Hoenen et al., 2006, Mateo et al., 2011, Reid et al., 2007, Schwarz et al., 2017, Watanabe et al., 2007, Xu et al., 2014, Zhang et al., 2012, Wan et al., 2017)
RNA-dependent RNA polymerase (L)
RNP complex component; homo-dimerizes; binds to genomic and antigenomic RNA, VP35, and VP30; mRNA capping enzyme
Genome replication and mRNA transcription; co-transcriptional editing
(Sanchez et al., 1996, Volchkov et al., 1995, Mühlberger et al., 1999, Groseth et al., 2009, Trunschke et al., 2013, Ferron et al., 2002, Tchesnokov et al., 2018, Shabman et al., 2013)
The viral envelope is derived from host cell membranes and is considered to have a lipid composition similar to that of the host-cell plasma membrane (Bavari et al., 2002, Feizpour et al., 2015). Ebolavirus glycoproteins are acylated (Ito et al., 2001).
The spike glycoproteins of ebolaviruses are highly glycosylated with N-linked high mannose, hybrid, and bi-, tri-, and tetra-antennary complex glycans with and without fucose and sialic acid and with O-linked glycans of the neutral mucin type. Glycans constitute >50% of the GP1,2 total mass. In contrast to marburgvirus GP1,2, ebolavirus GP1,2, may be strongly sialylated (Collar et al., 2017, Ritchie et al., 2010). Ebolavirus NP is O-glycosylated and possibly sialylated (Watanabe et al., 2006, Huang et al., 2002). sGP is N-glycosylated and C-mannosylated (Falzarano et al., 2007, Falzarano et al., 2006). ssGP is N-glycosylated (Mehedi et al., 2011). Δ-peptide is O-glycosylated and sialylated (Volchkova et al., 1999).
The ebolavirus genome has the gene order: 3′-NP-VP35-VP40-GP-VP30-VP24-L-5′ (Figure 2.Ebolavirus). The extragenic sequences at the extreme 3′- (leader) and 5′- (trailer) ends of the genome are conserved and partially complementary. Genes are flanked by conserved transcriptional initiation and termination (polyadenylation) sites. Most genes are separated by non-conserved intergenic sequences, but some genes overlap. Most of these overlaps are extremely short and limited to a highly conserved pentamer. In addition, most genes possess relatively long 3′- and 5′-noncoding regions (Sanchez et al., 1993, Ikegami et al., 2001, Sanchez and Rollin 2005, Towner et al., 2008). Similar to cuevaviruses, but contrary to marburgviruses, the GP genes of ebolaviruses possess three overlapping ORFs that can be joined through co-transcriptional polymerase stuttering (Sanchez et al., 1996, Volchkov et al., 1995).
Figure 2.Ebolavirus. Schematic representation of the ebolavirus genome organization. Genomes are drawn to scale. Courtesy of Jiro Wada, IRF-Frederick, Fort Detrick, MD, USA.
The replication strategy of ebolaviruses is highly similar to that of cuevaviruses and reminiscent of that of marburgviruses (Brauburger et al., 2015, Manhart et al., 2018). Ebolavirions enter host cells mainly by macropinocytosis (Aleksandrowicz et al., 2011, Nanbo et al., 2010, Saeed et al., 2010). Ebolavirus GP1,2 mediates cell surface attachment factor (e.g., C-type lectins, integrins, T-cell immunoglobulin and mucin domain 1 [TIM-1]) binding and subsequent low pH-dependent fusion into endosomes (Davey et al., 2017). Cathepsin L and/or B cleavage is required for GP1,2 binding to the endosomal receptor Niemann-Pick disease, type C1 (NPC1), which is also used by cuevaviruses and marburgviruses (Carette et al., 2011, Côté et al., 2011, Ng et al., 2014, Chandran et al., 2005, Schornberg et al., 2006, Misasi et al., 2012). Uncoating is presumed to occur in a manner analogous to that of other mononegaviruses. Ebolavirus transcription and genome replication take place in the cytoplasm and, in general, follow the models for members of the families Paramyxoviridae and Pneumoviridae. Transcription starts at the conserved transcriptional initiation site, and polyadenylation occurs at a stretch of uridine residues within the transcriptional termination site. The 5′-terminal non-coding sequences likely form hairpin-like structures in all (capped and polyadenylated) mRNAs. Replication involves the synthesis of full-length positive-sense genome copies (antigenomes) (Brauburger et al., 2015, Weik et al., 2002, Sanchez and Kiley 1987, Bray et al., 2000). During infection, massive amounts of nucleocapsids accumulate intracellularly and form intracytoplasmic inclusion bodies, which are the sites of ebolavirus transcription, replication, and nucleocapsid assembly (Hoenen et al., 2012). Mature nucleocapsids are transported for envelopment to the plasma membrane, where budding occurs in a VP40-mediated process (Bornholdt et al., 2013, Harty et al., 2000, Licata et al., 2003, Noda et al., 2002, Pavadai et al., 2018) (Figure 3.Filoviridae).
Ebolaviruses appear to be endemic in Western Africa (EBOV, TAFV), Middle Africa (BDBV, EBOV), Eastern Africa (BDBV, SUDV), and Eastern and South-eastern Asia (RESTV). The natural hosts of most ebolaviruses are unknown (bats are suspected for all; RESTV also naturally infects domestic pigs [Sus scrofa domesticus Erxleben, 1777]) and spread of ebolaviruses is not associated with any vector. The route of initial human infection is unknown (Amman et al., 2017). The major route of human-human transmission of filoviruses requires direct contact with blood, bodily fluids, or injured skin. Three ebolaviruses (BDBV, EBOV, and SUDV) are notorious and highly lethal human pathogens. TAFV has caused a single case of severe but nonlethal human disease, whereas Reston virus RESTV has thus far only caused inapparent human infections (Kuhn 2018, Albariño et al., 2017). In the laboratory, rodents (mice, guinea pigs, hamsters), nonhuman primates (common marmosets, crab-eating macaques, grivets, hamadryas baboons, rhesus monkeys), carnivores (domestic ferrets) and suids (domestic pigs) can be infected experimentally with various ebolaviruses, but lethal infection of rodents requires sequential adaptation (Siragam et al., 2018, St Claire et al., 2017, Marsh et al., 2011).
The antigenicity of ebolaviruses is primarily due to their spike glycoproteins. Numerous anti-GP1,2 monoclonal antibodies have been described that are specific for a particular ebolavirus or cross-reactive among ebolaviruses. Importantly, the ability of a monoclonal antibody to neutralize ebolavirus infection in vitro is not necessarily predictive of protective efficacy in vivo; vice versa, antibodies that are non-neutralizing in vitro have shown to be protective in vivo. Ebolaviruses and marburgviruses have only limited antigenic relatedness, and individual ebolaviruses can be differentiated with certain antibodies (Lee et al., 2008, Bramble et al., 2018, Dias et al., 2011, Flyak et al., 2018, Saphire et al., 2018, West et al., 2018, Wec et al., 2017).
Ebolavirus: from the Ebola (Legbala) River in Zaire/Democratic Republic of the Congo, where the first registered outbreak of Ebola virus disease occurred (Henry 2015).
PAirwise Sequence Comparison (PASC) using coding-complete ebolavirus genomes is the primary tool for ebolavirus species demarcation. Genomic sequences of ebolaviruses of different species differ from each other by ≥23% (Bào et al., 2017). Genomic features, such as number and location of gene overlaps, ebolavirus host and geographic distribution, and ebolavirus pathogenicity for different organisms are also taken into account for species assignment.
Phylogenetic relationships across the genus have been established from maximum likelihood trees generated using coding-complete or complete genome sequences (Figure 4.Filoviridae) or by analyzing filovirus L amino acid sequences (Wolf et al., 2018).
(Goldstein et al., 2018)
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