Bases Legales

Influenza viruses are a group of virus genera in the Orthomyxoviruses family of RNA viruses. This family includes three distinct influenza genera as well as a number of other virus genera. There are three distinct phylogenetic groupings of influenza viruses generally divided into influenza A, B and C. Of these influenza A viruses are the most significant regarding human health impact. Influenza A are the major source of numerous serious viral disease outbreaks including all known influenza pandemics that resulted in large numbers of human fatalities (Lam et al., 2008; Fukuyama and Kawaoka, 2011). In addition to occasional pandemics, influenza A causes annual epidemics which cause significant economic losses.

Furthermore, the virus may also cause outbreaks in poultry and other domestic animals, requiring costly and laborious countermeasures. Therefore, influenza virus has a substantial impact on health and the global economy (Edinger, Pohl and Stertz, 2014). Although in most cases infection with influenza A causes a severe but non-fatal disease, in patients with

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compromised immunity such as the elderly, young children and people suffering from chronic disease, influenza is associated with high mortality. In the United States, some estimates indicate that influenza infection is responsible for approximately 200,000 hospitalizations and 36,000 deaths in a typical endemic season (Taubenberger and Morens, 2008).

Influenza A are negative-sense, single-stranded, segmented RNA viruses and can be characterised based on the antigenic properties of their two primary membrane expressed glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (Tong et al., 2012). There are, at present, 18 different known H antigens (H1 to H18) and 11 different known N antigens (N1 to N11). HA is a protein that mediates binding of the virion to target cells and entry of the viral genome into the target cell, while NA facilitates the release of progeny virions from infected cells. Unlike the majority of other RNA viruses, influenza replicates within the nucleus of cells (Boulo et al., 2007; Hutchinson and Fodor, 2013; Martín-Benito and Ortín, 2013). Due to this method of replication, the virus must overcome or evade a number of additional barriers to replication when compared to RNA viruses that do not depend on entry to a cell nucleus. Viral entry is a dynamic process that requires the successful completion of several steps:

attachment to target cells, internalization into cellular compartments, endosomal trafficking to the perinuclear region, fusion of viral and endosomal membranes, “uncoating” and the subsequent import of the viral genome into the nucleus (Böttcher et al., 2006; Martín-Benito and Ortín, 2013). The influenza HA protein mediates both the attachment of the influenza virus to the target cell and the fusion process which allows the virus to pass its genetic material through the cell membrane. HA is a diverse protein and is the most significant factor in determining virus tropism (Sahini, Tempczyk-russell and Agarwal, 2010; dos Reis et al., 2011).

In general, for influenza replication HA is synthesized as an initial precursor molecule designated as HA0, this molecule is then subsequently cleaved into HA1 and HA2 by serine proteases (trypsin-like enzymes) for activation. These proteases work by recognizing a conserved Q/E-X-R motif found at the HA cleavage site as part of the HA maturation process (Böttcher-Friebertshäuser, Klenk and Garten, 2013). The receptor binding region of the HA molecule is located in the head region of the molecule with the stalk region mediating cell fusion (Iba et al., 2014). It is this fusion process that requires the cleavage of the HA molecule as this process allows the formation of the mature HA structure. Upon binding to the host cell the HA to undergo a low-pH-induced, irreversible conformational change in endosomes post-endocytosis that drives fusion of the HA into the host cell (Scott et al., 2012).

The discovery recently two novel influenza strains identified from bat hosts, have been designated H17N10 and H18N11 (Tong et al., 2012), has resulted in a rapid re-evaluation of many previously accepted theories. These novel viruses appear to have many unique traits

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that are not present in other influenza A strains (X. Sun et al., 2013; Juozapaitis et al., 2014).

The phylogenetic relationship between these different strains is displayed in figure 9. While the association between influenza viruses and wild birds has long been understood (J. F.-W.

Chan et al., 2013) the discovery of novel influenza virus in bats has resulted in a significant shift in the influenza research field. The discovery of the H17 and H18 strains in new world bats has cast doubt on the accepted paradigm that aquatic birds are the primary historical hosts of influenza A (Tong et al., 2012, 2013; X. Sun et al., 2013). While sialic acid is the dominant receptor in mediating the interaction between cell and virus the process of viral cell binding and entry may also involve numerous other receptors (Edinger, Pohl and Stertz, 2014).

While the exact mechanisms of these interactions are not yet fully understood and the relative roles are still matters for debate, the crystal structures of the newly discovered H17 and H18 indicate that these proteins do not bind sialic acid (Tong et al., 2013). This finding may indicate that the binding and entry mechanisms of influenza A viruses are more complex than previously thought.

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Figure 9: Influenza A phylogeny based on hemagglutinin subtype.

Showing distinct grouping of group 1 and 2 HAs' and highlighting the H17 and H18 novel subtypes

1.3.3. Bat borne influenza research

The identification of two novel influenza viruses has led to a rapid series of studies examining the characteristics of these new viral strains. Initial studies demonstrated that these strains did not bind to sialic acid receptors, a trait common to all other influenza A strains (X.

Sun et al., 2013) and the NA derivatives of these strains also do not demonstrate standard enzymatic activity common to other NA strains (Li et al., 2012). The unique features of the HA and NA of the H17 and H18 virus isolates indicate that these viruses do not bind to sialic acid as other influenza strains do (Tong et al., 2013) and to date, the receptor or receptors for these strains have not yet been identified.

Although the receptor/s remain unknown, significant analysis of the HA proteins of H17 and H18 viruses have been carried out both to determine the relationship these proteins have

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to other HA sequences and the potential for these viruses to become zoonotic (Mänz, Schwemmle and Brunotte, 2013; Freidl et al., 2015). Initial studies made use of bat influenza virus sequences in a reverse genetics system. However, these approaches failed to generate infectious virus surrogates (Juozapaitis et al., 2014; Zhou et al., 2014) when infection studies relied on the novel HA proteins. More recently the reconstruction of bat chimeric influenza virus particles demonstrated that all genomic segments of at least the H17N10 are capable of infecting specific cell lines (Juozapaitis et al., 2014). However intial studies failed to demonstrate the ability of HA and NA segments from H17 to produe functional products that allow viral entry and replication in mammalian cells (Juozapaitis et al., 2014). This study was however rapidly followed by a study demonstrated that the H17N10 proteins could, in fact, mediate cell entry (Hoffmann et al., 2016) but only within a limited number of cell lines. This study demonstrated only a single cell line identified as susceptible from the non-bat derived cell lines, and in fact, it was also determined that only 50% of tested cell lines derived from bats were, in fact, susceptible to the H17N10 expressing viral particles (Hoffmann et al., 2016).

Particularly interesting in this data is the fact that while transduction appeared to be efficient in three of the bat cell lines, it was not universal in all cell lines derived from bats.

Where infection did occur is was in two Miniopterus sp. and two Pteropus sp. derived cell line.

This finding suggests that these viruses may be restricted to a particular set of closely aligned species as the Miniopteridae and Pteropidae bat families are closely related to the Phyllostomidae family from which the original H17 and H18 samples were isolated (Agnarsson et al., 2011; Tong et al., 2012). However, this also leads to further questions as the cell line derived from Pteropus dasymallus yayeyamae failed to facilitate transduction as did the cell line derived from Rousettus leschenaultii and as both of these bats species are also members of the Pteropus genus the pattern of susceptibility of species to these new influenza viruses are apparently not simplistic. Finally, as these cell lines are relatively newly derived and immortalised (Maeda et al., 2008; Maruyama et al., 2014) detailed characterisations of their surface proteins and proteases are not yet available, making conclusions drawn from H17 PV infection of these cells more complex.

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Table 4: Cell lines used in successful VSV based pseudotyping of H17 and H18 viruses. Underlined entries are cell lines originating from bats. Bold entries are those cell lines which were found to be permissive for viral transduction (Maruyama et al., 2016). * Maruyama et.al. did not report the specific variety of MDCK cells used in this study, see section 6.4. for further details. ¥ indicates cells extracted from bats.

CELL LINE SPECIES ORIGIN

VERO E6 Chlorocebus sp. Kidney

HEK293 Homo sapiens Kidney

MDCK* Canis lupus familiaris Kidney

SK-L Sus scrofa Kidney

QT6 Coturnix japonica Muscle

BKT1 Rhinolophus ferrumequinum¥ Kidney

FBKT1 Pteropus dasymallus yayeyamae¥ Kidney

YUBFKT1 Miniopterus fuliginosus¥ Kidney

INDFSPT1 Pteropus giganteus¥ Spleen

DEMKT1 Rousettus leschenaultii¥ Kidney

ZFBK11-97 Epomophorus gambianus¥ Kidney

SUBK12-08 Miniopterus schreibersii¥ Kidney

ZFBS13-75A Eidolon helvum¥ Spleen

The susceptibility of the MDCK cells is less clear and showed far lower levels of successful transduction (Maruyama et al., 2016), this also does not agree with a previous study that showed H17 HA failed to bind to MDCK cells (X. Sun et al., 2013). While the reasons for this are currently unclear, it has been hypothesized that low affinity of H17 HA for the MDCK cells may mean that the level of binding is below the threshold of detection for the assays used in this earlier study (Maruyama et al., 2016). However, another hypothesis that may resolve this discrepancy is that since the MDCK cell line is highly diverse. Many different strains of MDCK cells available, including the parental line (designated ‘NBL-2’ ATCC^® cat # CCL-34™Ψ, MDCK I (EEACC cat # 00062106), MDCK II (EEACC cat# 00062107), MDCK.1 (ATCC^® cat#

CRL-2935™Ψ, MDCK.2 (ATCC® cat# CRL- CRL-2936™Ψ, super dome (ATCC^® cat # CRL-2286^™) and super tube (ATCC® cat# CRL-2285™Ψ cell lines as well as several other strains (Dukes et al., 2011). It is possible that studies such as those investigating cell susceptibility to the H17 virus may, in fact, be comparing different variants of MDCK and so differences in the observed susceptibility of the respective MDCK cells would not be surprising.

Although the ‘live’ H17N10 and H18N11 viruses have not been isolated and so their potential for future zoonosis is still unknown the development of the replication-incompetent VSV pseudotype system allows the tropism of these viruses to be studied. The further

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development of such tools coupled with the potential generation of chimeric HA constructs may allow detailed study of the both the potential for zoonosis of these viruses and also the potential for the generation of antiviral interventions in particular to the well conserved stalk region (Krammer et al., 2014).