Problemas Secundarios

The Filoviridae are negative-sense, single-stranded RNA viruses that have a filamentous morphology and are linked to outbreaks of viral haemorrhagic fever. Viruses in this family include Ebolavirus and Marburgvirusand Lloviu virus (LLOV), of these all members of the Ebolavirus and Marburgvirus have been linked to infection of humans while LLOV has only been isolated from bat carcasses (Leroy, Gonzalez and Baize, 2011; Negredo et al., 2011). Table 3 displays the known members of the Filoviridae family and highlights the virus species that have previously been responsible for human infections. The Filoviridae are characterized by a sudden epidemic occurrence, and well as a high lethality, both traits are often considered clear indications of spillover events (Mathews, 2009). Although the reservoir host for any species of Ebolavirus has not been conclusivly determined, there is evidence of a bat origin for these viruses (Spengler et al., 2016). IgG-specific to the Zaire Ebolavirus strain (ZEBOV) antibodies have been detected in the fruit bats Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata during the Ebolavirus of 2001 and 2005 in Gabon and the Republic of the Congo (Pourrut et al., 2007).

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Table 3: List of currently accepted members of the Filoviridae family of viruses with details of the year of first identification, Reservoir host, if known, and recorded human outbreaks of the disease.

GENUS SPECIES RESERVOIR HOST

et al., 2011) Miniopterus schreibersii 2011 n/a

MARBURGVIRUS

Marburg marburgvirus

Rousettus aegypti

(Towner et al., 2009) 1967 1967, 1975, 1980, 1987, 1998, 2005, 2007, 2008

Ravn virus Rousettus aegypti (Towner

et al., 2009) 1996 1987, 1998, 2007

EBOLAVIRUS

Reston ebolavirus Multiple Bat sp.(Jayme et

al., 2015) 1990 n/a

Sudan ebolavirus

Unknown

1998 1976, 1979,2000, 2004, 2011, 2014

Ta¨I Forest ebolavirus 1995 1994

Zaire ebolavirus 1976

1976, 1977, 1994, 1995, 1996, 2001, 2002, 2003, 2004, 2007, 2008, 2014

Bundibugyo ebolavirus 2008 2007, 2012

The Filoviridae genome encodes seven structural proteins (Figure 7), and in addition, the Ebolavirus genome encodes a further two non-structural, soluble, glycoproteins (GP):

usually designated a soluble GP (sGP) and small soluble GP. The Marburgvirus consists of two species termed Lake Victoria Marburg Marburgvirus (MARV) (sometimes refered to as Lake Victoria Marburg virus) and Ravn virus (RAVV) (Burk et al., 2016). Ebolavirusis a more diverse group with five currently accepted different species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Taï Forest virus (TAFV), Reston ebolavirus (REBOV) and Bundibugyo ebolavirus (BEBOV) (Towner et al., 2008). In addition, novel Filoviridae was identified in European bats in 2011; this novel Filoviridae designed Lloviu virus appears to be more distanctly related to either Marburgvirus or Ebolavirus then these viruses are to each other (Negredo et al., 2011), and is not known to cause disease in humans.

As indicated in table 3 the majority of Filoviridae outbreaks have been linked to species of Ebolavirus; this includes the most recent 2014 outbreak which was linked ZEBOV and resulted in a greater number of human infections then recorded in all other Filoviridae outbreaks in total (Na et al., 2015). However, there is significant variation in the apparent pathogenicity of the Ebolavirus species in humans; ZEBOV is the most pathogenic (up to 90%

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case fatality rate), followed by SEBOV (approximately 50% case fatality rate) and BEBOV (approximately 40% case fatality rate). TAFV and REBOV cause lethal infections in nonhuman primates but have not been associated with fatal infections in humans under natural conditions.

Figure 7: Schematic representation of the seven structural proteins encoded by the Filoviridae genome.

Proteins are nucleoprotein (NP), Polymerase cofactor vp35 (vp35), viral matric protein (vp40), glycoprotein (GP), transcription activator vp30 (vp30), matric protein vp24 (vp24) and the RNA polymerase (Poly). Figure produced in inkscape with reference to (Burk et al., 2016)

Of particular importance in the study of Filoviridae is the role played by the glycoprotein (GP), this protein is responsible for cellular attachment and entry (Sanchez et al., 1996). The first step in viral replication is the viral attachment mediated by the GP which binds to cellular receptors is followed by endocytosis (Wool-lewis and Bates, 1998).

The GP protein of the Ebolavirus is typical of the Filoviridae in terms of function, however, in Ebolavirus GP correct protein functioning requires post transcriptional editing, to express structural GP (Volchkov et al., 1998). In addition, to the strucutural GP, the Ebolavirus GP gene also encodes two non-structural GP proteins, sGP and ssGP that are not present in other Filoviruses (Khun, 2008). The three GP proteins which are encoded by the fourth gene of the Ebolavirus genome are the full-length 676-residue surface glycoprotein (GP), the 364-residue secreted glycoprotein (sGP), and the 298-364-residue small secreted glycoprotein (ssGP).

The GP is produced by ribosomal slippage or stuttering by the viral polymerase at the editing site (seven consecutive template uridines) in the GP gene (de La Vega et al., 2015). This slippage adds an eighth adenosine residue to the transcribed mRNA and allows for the fusion of the two separate ORFs of this gene, this in turns produces a structural GP composed of two subunits GP1 and GP2 (Sanchez et al., 1996). When transcribed without the ribosomal

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slippage affect the unedited mRNA transcripts produce sGP. Furthermore the addition of two additional adenosine residues during this process (i.e. nine adenosines) produces ssGP (Dolnik et al., 2015). After the sGP RNA is translated, the immature sGP is cleaved by cellular furin protease to produce the mature sGP (Lee and Saphire, 2009). The precise function of this non-structural GP is unknown, however, is has been observed that large amounts of sGP are produced and secreted into the blood of Ebolavirus patients (Sanchez et al., 1996). It has also been demonstrated that antibodies present in serum extracted from Ebolavirus convalescent patients show greater affinity for sGP over GP (Maruyama et al., 1999). It is, therefore, possible that the sGP has a role in immune evasion by binding to antibodies that would otherwise bind to the GP. The ssGP produced by the post transcriptional inserting of two adenosine bases is poorly understood at present. To date the role this protein plays in viral replication and pathogenesis is unknown (Volchkova et al., 2015).

The GP of Ebolavirus facilitates cellular binding and fusion with the host cell; this protein is produced by the post transcription insertion of an additional adenosine at hairpin loop (Sanchez et al., 1996) that results in a frameshift causing the production of protein 676-residues in length (677 amino acids in REBOV). This protein is type I transmembrane GP that is the immature form of the final Ebolavirus GP (Sanchez et al., 1996). Due to the encoding of both proteins by the same gene GP and sGP have an identical 295 amino acid N-terminus, but differ at C-termini (Lee and Saphire, 2009). The differences in the proteins C-termini change the disulphide bonding and yield different final structures for the proteins. The GP protein is a trimer while the sGP forms a dimer. The immature Ebolavirus GP is cleaved by furin at a multi-basic site, forming two subunits designated GP1 and GP2. These subunits are connected via a disulphide linkage between Cys53 of GP1 and Cys609 of GP2 (Lee and Saphire, 2009; Volchkova et al., 2015) (figure 8). The finished GP protein comprised of two linked subunits then forms trimers on the surface of the assembling virons. Virons then escape the cell via the cleavage of the GP through the action of a NF- -converting enzyme (TACE) allowing the virion to detach from the cell membrane, thus releasing the viron from the cell (Dolnik et al., 2004). Figure 8 describes the basic composition of the Ebolavirus GP gene and highlights key protease cleavage locations.

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Figure 8: Structure of the Ebola virus Glycoprotein showing domains typical for the Filoviridae.

GP1 domain is divided into head fragments indicated in blue, base fragments indicated in green. Glycan C indicates the glycan cap; Mucin D indicates the mucin-like domain; RBD is receptor binding domain; SP is a signal peptide. GP2 region highlights the following domains HR1 and HR2 are heptad repeats 1 and 2 respectively; MPER, membrane-proximal external region and TM, indicates the transmembrane domain, The final structure Cyto. Tail indicates the cytoplasmic tail segment, Figure produced with inkscape with reference to (Lee and Saphire, 2009; Bale et al., 2012)

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When an Ebolavirus viron comes into contact with a cell, the GP is the protein responsible for both binding and fusion to allow entry of virus into the cell. Initial binding of the GP to host cell receptors is mediated by the RBD region in the GP1 domain of the protein (figure 8). However, virus cell interaction in filoviruses is thought to differ from many other enveloped viruses as in filoviruses amino acid residues do not appear to directly interact with a cell surface receptors (Moller-Tank and Maury, 2015). The initial binding of these viruses to cells are instead mediated through non-specific receptors: C-type lectins (CLECs) that interact with glycans on Ebolavirus GP and PtdSer receptors that interact with the viral envelope (Moller-Tank and Maury, 2015). The interaction between the virus and these relatively non-specific cellular proteins leads to uptake of the viron by the cell via Macropinocytosis (Mingo et al., 2015). Macropinocytosis is a form of clathrin-independent endocytiosis (Lim and Gleeson, 2011). Viral entry is made more efficient by the activity of the mucin-like domain and glycan cap both of which mediate GP binding to several cell surface proteins (Simmons et al., 2003; Martinez et al., 2011; Takada, 2012). After virus endocytosis, the GP is cleaved by intrinsic host proteases that remove 80% of the mass of the GP1 subunit, including the mucin-like domain and glycan cap (Côté et al., 2011; Martinez et al., 2012). After cleavage of GP in the endosome, the receptor-binding sites on GP become exposed, and the GP1 head then is able to bind to its receptor, Niemann-Pick C1 (NPC1) protein (Carette et al., 2011; Chandran et al., 2005; Côté et al., 2011). Subsequent conformational changes in GP facilitate fusion between viral and endosomal membranes.

In addition, to the role GP plays in cell entry, there is a growing number of studies that have linked this protein to pathogenic differences among viral species. Studies comparing the pathogensis of different Ebolavirus species have shown evidence that ZEBOV GP causes endothelial cell damage in both human and monkey vessel explants, while REBOV appears asymptomatic in humans while being detrimental to monkey cells (Takada, 2012). ZEBOV and REBOV also significantly differ in their susceptibility to furin cleavage (Lee and Saphire, 2009;

Dolnik et al., 2015) and amino acid sequence at the TACE cleavage site (Dolnik et al., 2004).

Moreover, it has been reported overexpression of GP causes cytotoxicity and downregulation of a number of cellular surface proteins, including several involved in cellular adhesion ( ¹ -integrin, ⁵-integrin and ^V-integrin) and immune surveillance (MHC class I), but that lower levels of GP expression do not display the same the cytotoxic effects (Alazard-Dany et al., 2006).

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1.3.3.1. Filoviridae antiviral and vaccine development

Ebolavirus is the most well studied of the Filoviridae; this is largely due to the increased frequency of human outbreaks of this virus compared to other filoviruses. Ebolavirus can be considered a stage 3 virus (Wolfe, Dunavan and Diamond, 2007) (Section1.1), although REBOV has not been recorded as a human infection other species of Ebolavirus do fall into this category. Sporadic outbreaks of this virus (Table 3) have been linked wild animals, and the virus has not proven capable of efficient human-human spread (Carroll et al., 2015; Na et al., 2015).

The 2014 Zaire Ebola outbreak in West Africa and has resulted in an estimated 10,500 laboratory confirmed Ebola cases, that resulted in more than 4,800 confirmed deaths (World Health Organization, 2016). Prior to this, Ebola outbreaks were reported on average every 1.5 years since the viruses first discovery in 1976, with the largest previous outbreaks infecting 425 people (Martinez et al., 2012). The unprecedented scale of this latest outbreak has led to a rapid increase in the pace of research. Much of the research has, by necessity, focused on the development of effective treatments and vaccines.

The GP of Ebolavirus as in other filoviruses is the only viral envelope protein and the only surface accessible protein on the Ebolavirus virion. Thus this protein constitutes the sole target for the neutralizing antibody response (Yu et al., 2015). As such, much of the research has focused on identifying the most antigenically active domains in GP and the generation of drugs targeting regions essential for cellular entry that can be targeted (Hofmann-Winkler, Kaup and Pöhlmann, 2012).