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Symposium Report |
1 Institute of Molecular Medicine and Cell Research, Albert Ludwigs University, Freiburg im Breisgau, Germany 2 Kantonsschule Hohe Promenade, Promenadengasse 11, 8001 Zurich, Switzerland 3 Institute for Infectious Diseases, University of Bern, Bern, Switzerland
Abstract
A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application.This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used.
(Received 20 September 2004;
accepted after revision 15 October 2004; first published online 12 November 2004)
Corresponding author C. Rhême: Institute of Molecular Medicine and Cell Research, Albert Ludwigs University, Stephan-Meier Strasse 17, D-79106 Freiburg im Breisgau, Germany. Email: crheme{at}hotmail.com
Semliki Forest virus (SFV) and Sindbis virus (SIN) are among at least 24 species in the alphavirus genus of the family togaviridae. They are enveloped single-stranded, positive-sense RNA viruses that replicate entirely in the cytoplasm without any DNA intermediate. Their genomes are encapsidated in a network of the unique capsid protein, forming the nucleocapsid of the virion particle. Structurally, mature virions resemble a small sphere (70 nm in diameter) coated with a host cell-derived lipid bilayer originated by budding. Eighty glycoprotein spikes project out of this envelope. Alphaviruses have a very broad host range and replicate to high titres in many cells of both vertebrates and invertebrates (Strauss & Strauss, 1994). Recently, some alphaviruses have received considerable attention as gene-delivery vehicles. Full-length replication-competent clones, self-replicating packaging-defective RNA vectors (replicons) as well as DNA-based vectors have been developed from SFV (Liljestrom & Garoff, 1991), SIN (Xiong et al. 1989) and Venezuelan equine encephalitis virus (Davis et al. 1989).
Biology of alphaviruses
Genomic organization. The single-stranded, around 12 kb long genomes of SFV and SIN are divided into two open reading frames (ORF). The first ORF encodes four non-structural proteins, designated nsP1 to nsP4, responsible for transcription and replication of viral RNA. The second ORF, under the control of a 26S subgenomic promoter, codes for the structural proteins required for the encapsidation of the viral genome and the proper assembly into enveloped particles. They include the capsid protein, the glycoproteins E1, E2 and E3 and the 6K protein. The structural proteins are not necessary for viral replication, but are required for virus propagation, together with the packaging signal located in the coding region of nsP2 in SFV and of nsP1 in SIN (Strauss & Strauss, 1994).
Life cycle. After attachment of the virion to a cell receptor via the exposed E2 portion of the spikes, the virion enters endosomes. The release of the nucleocapsid into the cytoplasm occurs upon fusion of the viral and endosomal membranes as a consequence of conformational changes in the glycoprotein spikes induced by the endosomal acidic environment. After dissolution of the nucleocapsid, the liberated genomic RNA codes for the polyprotein of non-structural proteins. Its sequential processing by the cysteine protease activity of nsP2 determines the specificity of the replication complex. Initially, the primary complex synthesizes the negative-sense RNA, using the original genomic RNA as a template. Later, upon assembly of a complex composed of the four individual proteins, minus-strand RNA synthesis ceases and the minus-strand RNA is itself used as a template for the transcription of full-length genomic plus-strand RNA as well as of positive-sense subgenomic (26S) RNA. The latter is translated into a polyprotein, composed of the structural proteins. Using its serine protease activity, the capsid autocatalytically cleaves itself off and the remaining polypeptide is targeted to the lumen of the endoplasmic reticulum, where cellular enzymes will further process it into the three glycoproteins and the 6K protein. After circulating through secretory vesicles, the mature glycoproteins ultimately localize to the plasma membrane, where they interact through their cytoplasmic domain with the newly formed nucleocapsid. This interaction leads to the release of mature viral particles upon budding from the plasma membrane (Strauss & Strauss, 1994).
Host specificity. Alphaviruses infect cells by a receptor-mediated mechanism. Amongst others, major histocompatibility complex I (MHC I) and high-affinity laminin receptors have been proposed as candidates (Helenius et al. 1978; Wang et al. 1992). Apart from infecting neurones in the CNS or in primary cultures, the neurotropic SFV or SIN or their vector derivatives have an impressively broad host range in cell culture systems, infecting a wide variety of mammalian primary or immortalized cell lines, as well as non-mammalian ones (for a detailed review, see Lundstrom, 2003). This makes SFV and SIN powerful vector tools to deliver genes in otherwise poorly transfectable cells or in non-dividing cells not prone to be infected by other viral-based vectors.
Virulence and pathogenicity. SFV and SIN have been extensively used in mice and rats as a model for viral neuropathogenesis and neurodegenerative diseases (multiple sclerosis). Following infection by intraperitoneal or intranasal inoculation, virulent strains of SFV, such as the original isolates L10 and SFV4, multiply rapidly in neurones and cause lethal encephalitis. Infections with less virulent strains, such as A7 and M9, generally kill only newborn mice. In adult mice, avirulent virus multiplication leads mainly to CNS demyelination and clearance. Clearance usually occurs around 7–10 days after infection. Of special interest is the use of SJL mice, which display a defect in innate expression of the immune response. In these mice, the infection-induced immune-mediated demyelination persists up to 1 year, and is not linked to virus persistence (Donnelly et al. 1997; Fazakerley, 2002). Similar in some aspects to experimental autoimmune encephalomyelitis, infection of SJL mice with avirulent strains of SFV mimics a model of human multiple sclerosis.
The determination of virulence has been localized to different regions of the genome, including the 5'UTR, the glycoprotein E2 and the different nsPs. Virulent and avirulent strains of SFV differ in their ability to multiply in neurones and rapidly spread in the CNS. In the case of virulent strains, a lethal threshold is reached before the immune system can intervene. Interestingly, it was recently reported that mRNA expression of proinflammatory cytokines in the CNS during SFV infection correlates solely with viral replication rate. Neither qualitative nor quantitative differences in the levels of proinflammatory cytokines could be seen between different avirulent viruses, carrying protein structures that originated from either the highly neurovirulent SFV4 clone or the attenuated A7(74) parental virus (Tuittila et al. 2004). Virulence of neurotropic viruses may, however, be relatively independent of the cytokine patterns they induced. For example, the lethal NSV variant of SIN and the clinically apathogenic SIN TE12 strain both induce similar patterns of cytokine mRNA in mice (Wesselingh et al. 1994). Attenuated strains, produced by point mutations in the nsP coding region which reduce the rate of replication, are considered to be the basis for the development of new, less cytotoxic and non-cytotoxic vectors.
Cytotoxicity. In the CNS, SFV infects both neurones and oligodendrocytes (Atkins et al. 1999). Neuronal fate depends on the virulence of the strain and the maturity of the infected neurone: while immature neurones allow productive replication of both avirulent and virulent strains and always succumb by apoptosis, mature neurones are only permissive for virulent strains and eventually die by necrosis (Glasgow et al. 1997). Accordingly, lethal virulent and avirulent infections of neonatal mice are always associated with extensive induction of apoptosis in immature neurones. In an adult rat model of intranasal infection with avirulent A7 and virulent SFV4 strains, apoptosis was documented in the rostral migratory stream, where neuronal precursor cells migrate from the proliferating subependymal layer into the olfactory bulb. In addition, the virulent SFV4 strain provokes extensive areas of necrosis in the superficial layers of the olfactory bulb and in contiguous groups of more mature cortical, thalamic and hippocampal neurones (Sammin et al. 1999). Thus, cellular maturation processes provide a basis for the different susceptibilities of neuronal subpopulations towards alphavirus infection.
Semliki Forest virus-infected oligodendrocytes undergo apoptosis in mixed glial cell cultures (Glasgow et al. 1998). In vivo, infected oligodendrocytes can be damaged but recover and carry out remyelination after avirulent SFV infection. Alternatively, in the case of extensive irreversible damage to oligodendrocytes, the putative involvement of apoptosis remains unclear. Their death could be the consequence of either a direct induction of apoptosis by viral infection or of their targeting by cytotoxic T-cells recognizing viral antigens on their surface.
Death mechanisms. Because cytotoxicity offers advantages and disadvantages in the use of alphaviruses as vector tools, a good understanding of the molecular events responsible for the cellular demize of infected cells would be useful. Despite the tremendous number of published articles focusing on this aspect, it is still difficult to have a clear overview of the death mechanisms. Comparisons are somewhat difficult because infections were performed in many different cellular systems. Apart from necrosis, regulated death mechanisms are probably multifactorial, demonstrating interconnections among disparate signalling pathways, such as apoptosis, antiviral responses, ER stress and probably inflammation, though this remains to be proven.
Although the glycoproteins may be necessary for the complete cytopathic effect during SFV/SIN infection (Frolov & Schlesinger, 1994), it is generally admitted that the non-structural region of the viral genome is necessary and sufficient for the induction of apoptosis, while the structural region may be deleted without affecting apoptosis (Liljestrom, 1994). We favour active viral RNA replication as the trigger for cell death.
The presence of viral double-stranded RNA intermediates initiates the antiviral response, and infection leads to the inhibition of host protein synthesis. Interestingly, point mutations in the nsP2 gene attenuate or suppress both the cytotoxicity and the shut-off of host protein synthesis, delineating possible links between viral replication, antiviral defense and suicide of the host. The activation of the double-stranded RNA-activated protein kinase (PKR) has been proposed to contribute to both the blockage of protein synthesis and the induction of apoptosis (Terenzi et al. 1999; Balachandran et al. 2000). Very recently, Gorchakov and coworkers demonstrated that PKR-dependent and -independent mechanisms are involved in translational shut-off during SIN virus infection, but detailed investigations of their relations to mechanisms of cell death and virus persistence were not explored (Gorchakov et al. 2004).
B-cell Lymphoma/Leukemia 2 (Bcl-2) family members have been proposed to be involved in the regulation of alphavirus-induced apoptosis, but their effect is probably dependent on the cellular context: while overexpression of prosurvival members leads to persistent infection or at least in a delayed cell death in some cell lines, alphaviruses have been shown to overcome the protection in others, where Bcl-2 could be cleaved and inactivated by a caspase-dependent mechanism (Levine et al. 1993; Scallan et al. 1997; Grandgirard et al. 1998). Heterologous expression of Bcl-2 or Bcl-2 related protein (Bcl-XL) in the vector may ameliorate the outcome of infected cells. Conversely, the SFV-driven expression of Bcl-2 associated x protein (Bax) potentiates the cytotoxicity of the vector (Murphy et al. 2001). Being expressed in the CNS to a higher degree than Bcl-2, prosurvival Bcl-XL or Bcl-WEHI (Bcl-w) may be more likely to influence the restriction of neuronal damage (Sammin et al. 1999). Nevertheless, killing of mature neurones by virulent strains is more related to necrosis, and thus unlikely to be regulated by Bcl-2 family members.
Apart from being major key players in the execution of the apoptotic programme, caspases have also been involved in the more upstream initiation phase. Depending on the nature of the stimulus, caspases have been ordered in three classical pathways: the death-receptor, the mitochondrial and the ER stress pathways. As documented in Fig. 1, infection of HeLa cells by SFV induces the release of cytochrome c into the cytosol, as well as the activation of caspase-3 in a time-dependent manner. We are currently using recombinant SFV viruses carrying various genes that selectively interfere with the different death signalling pathways to identify the molecular mediators activated upon SFV infection. The simultaneous expression of green fluorescence protein (GFP) under the control of a second subgenomic promoter allows for the identification of infected cells.
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In 1989, the original creation of an alphaviral self-replicating and packaging-deficient expression vector (replicon; Xiong et al. 1989) was the starting point of the rapid development of various alphaviral vectors. Xiong et al. (1989) modified an infectious SIN virus by replacing the structural protein gene region with the chloramphenicol acetyltransferase (CAT) gene. In order to package the replicon RNA into particles, a helper SIN virus was used to supply the structural proteins. Moreover, by introducing a mutation in the non-structural gene region of the replicon that rendered it temperature-sensitive, Xiong et al. (1989) highlighted the promising potential of the system.
Full-length infectious vectors. An infectious clone containing the full genome of SFV was created in 1991 (Liljestrom et al. 1991). Later, SIN (Hahn et al. 1992) and Venezuelan Equine Encephalitis virus (Frolov et al. 1993) were engineered to contain a second subgenomic promoter upstream or downstream of the complete structural protein-coding region, allowing high level expression of heterologous genes. Recently, a novel replication-competent, double-subgenomic SFV vector derived from the avirulent A7(74) strain (Tuittila et al. 2000) was constructed (Vähä-Koskela et al. 2003). Containing the entire genome, these vectors share the common features of being fully infectious. Although this is an advantage in vitro for increased protein production, their applications in vivo may be limited for safety reasons.
Replicon vectors. From at least three different alphaviruses, replicon expression vectors have been engineered from virus strains. In those vectors, the genome for the viral structural proteins has been replaced by a multiple cloning site. They retain the entire non-structural region as well as the natural subgenomic promoter. Packaged alphavirus-like particles are produced by cotransfection of in vitro-transcribed replicon RNA and a helper RNA (Liljestrom & Garoff, 1991; Bredenbeek et al. 1993).
Productive replication and high level expression of foreign genes can be initiated either by transfection of the genomic RNA into the cytoplasm of the cell or by its infection with packaged alphavirus-like particles. The system is self limiting because helper RNAs, which lack the packaging signal, are not encapsidated. Thus, replicons are single-cycle vectors incapable of spreading from infected to non-infected cells. The major drawback of this system is the risk, although at very low probability, of recombination between the two vectors, leading to the generation of wild-type genomes (Weiss & Schlesinger, 1991). To reduce the amplification of replication-competent viruses, further manipulations of the helper plasmid have been engineered. One approach renders the system conditional by necessitating a proteolytical activation of the glycoproteins (pSFV-Helper2; Berglund et al. 1993). In another approach, the structural genes are split onto two separate helper vectors (Frolov et al. 1997; Smerdou & Liljestrom, 1999).
DNA-layered, cytomegalovirus (CMV) or RNA polymerase II promoter. A further development of the alphavirus expression system is the so-called ‘DNA/RNA-layered vector’, where the viral cDNA sequence has been put under a eukaryotic promoter, such as the CMV immediate early promoter (Dubensky et al. 1996; Berglund et al. 1998; Kohno et al. 1998). The approach results in a layered expression system: after transfection of the DNA into the cell, the cellular RNA polymerase II transcribes the recombinant construct. Upon transport of the viral mRNA from the nucleus into the cytoplasm, the translated alphavirus replicase amplifies the viral RNA. This strategy offers the highest safety, owing to the absence of the genes coding for the structural proteins, which eliminates the risk of producing replication-proficient viruses. It also suppresses the need for expensive in vitro RNA transcription. These vectors are most adequate for in vivo applications, where transient, high-level protein expression is wanted, such as for recombinant vaccines (see below).
Less cytopathic and non-cytopathic vectors. As described above, replication of both naïve and recombinant SFV induces cell death. This limits the use of the vectors to transient expression studies. To broaden the range of applications, new SFV vectors containing one or several mutations in the nsP genes have been engineered. For example, the SFV(PD) vector, bearing two point mutations in nsP2, exhibits decreased cell toxicity, probably because of on-going host cell protein synthesis (Lundstrom et al. 2003). For this vector, viral replication is also reduced. However, exogenous proteins introduced by SFV(PD) are still robustly expressed.
Temperature-sensitive vectors. A further development of the SFV system was made possible by specific mutations in the non-structural proteins 2 (nsP2) and 4 (nsP4), which rendered the virus also temperature sensitive. Based on SFV(PD), the triple mutant vector SFV(PD713E) was engineered by adding a point mutation in amino acid position 713 of nsP2. The result was a less cytotoxic vector with prolonged transgene expression and host cell survival at 31°C (over 20 days), as compared to SFV(PD) (Lundstrom et al. 2003). The introduction of other nsP2 and nsP4 point mutations into SFV(PD) also led to vectors (e.g. SFV(PDE153) and SFV(PDTE)) with temperature-dependent gene expression (Lundstrom et al. 2001). Transgene expression by all these vectors occurs at 31°C (permissive temperature) but is limited or absent at 37°C (non-permissive temperature). Because of the lower permissive temperature, the application of the present temperature-sensitive vectors to studies in mammalian cells is limited to in vitro applications. Interestingly, SFV(PDE153) lead to interneurone-specific transgene expression in rat hippocampal slices cultured at 36°C, whereas both interneurones and pyramidal neurones were transduced at 31°C (Lundstrom et al. 2001). These results show that SFV vector mutants can be useful for the selective transduction of inhibitory neurones from the rat CNS.
Avirulence based on a temperature-dependent switch in host cell preference. In contrast to wild-type SFV, which mainly infects neurones (Ehrengruber et al. 1999) and causes lethal encephalitis (Griffin, 1998), the SFV strain A7 and its derivative A7(74) are avirulent in adult rodents, triggering only limited CNS infection (Bradish et al. 1971; Pusztai et al. 1971; Fazakerley et al. 1993; Oliver et al. 1997). The full A7(74) genome was cloned, and nsP1–4 (rather than the structural genes) identified as genetic determinants for avirulence (Tuittila et al. 2000). Based on the A7(74) cDNA, we generated two expression vectors: (i) VA7-EGFP, a fully replication-competent virus expressing the GFP reporter gene downstream from a second subgenomic promoter (Vähä-Koskela et al. 2003); and (ii) the replicon SFV(A774nsP)-GFP, containing the GFP transgene instead of the structural genes (Ehrengruber et al. 2003). Because our previous experiments had shown that nsP1–4 mutations can confer temperature dependence (see section on Temperature-sensitive vectors above), we tested the A7(74)-based recombinants at both 31 and 37°C in dissociated cell and tissue cultures from the rat CNS (Table 1; Ehrengruber et al. 2003). Interestingly, in more mature cultures at 37°C, both VA7-EGFP and SFV(A774nsP)-GFP transduce glial cells rather than neurones, whereas at 31°C prominent neuronal transduction occurs. In hippocampal slices prepared from neonatal rats and infected at 1 day in culture, by contrast, neurones are also transduced at 37°C, which is compatible with the fact that A7 and A7(74) are avirulent in adult but not neonatal and young rodents. The data show that, in addition to the developmental age, the temperature determines which CNS cell type becomes transduced, suggesting that A7(74) is avirulent in adult animals because it does not readily replicate in mature neurones at body temperature, while it still does so at lower temperatures. In addition, to our knowledge, our results show for the first time that temperature can alter the host cell preference of a virus.
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In vitro and in vivo protein expression. Alphavirus vectors are widely used in basic research for structural studies of proteins because they produce robust protein expression. Intracellular, secreted and membrane proteins as well as receptors have been successfully produced (extensively reviewed by Lundstrom (2003)). Although the system is very efficient for the production of proteins, the use of original vectors for most functional studies is restricted by the inhibition of host cell protein synthesis. The emergence of novel vectors combining reduced cytotoxicity and prolonged transient transgene expression should alleviate this problem for both in vitro and in vivo therapeutic applications.
Vaccine development. ‘Naked’ RNA (Zhou et al. 1994) and DNA vaccines (Berglund et al. 1998) have been developed and proven to provoke high immunogenicity and low antivector immunity. Alphaviral DNA vaccines exploit the above-described DNA/RNA layered replicons and are very attractive for several reasons: (i) compared to other viral vectors (poxvirus and adenovirus), there is a general lack of pre-existing immunity in the population; (ii) their expression is both transient and lytic, which circumvents the biosafety risks of chromosomal integration and the induction of immunological tolerance; (iii) the absence of viral structural proteins eliminates both the induction of an otherwise strong immune response towards the vector and the risk of amplification of recombined replication-competent viruses; (iv) they can be applied to therapeutic vaccination by the expression of a ‘self’-antigen to break tolerance and provide immunity to tumours as well as to viral, bacterial and protozoan organisms, for prophylactic and therapeutic purposes; and (v) they have been reported to be more efficient than other viral or conventional DNA vectors, requiring 100- to 1000-fold smaller DNA amounts per immunization (Leitner et al. 2000). The molecular reasons for the high vaccination efficiency, however, are not clear and have been disputed. Although it is accepted that this efficiency correlates with the virus-induced cell death, the opinions and rationales differ, as follows. Leitner et al. (2003) consider that the DNA delivery is not specifically targeted. The immunogenicity and efficacy of SIN DNA vaccines is thought to be improved significantly by delivering stronger ‘adjuvant-type’ signals to the innate immune system, through activation of antiviral pathways in transfected cells or by the presentation of apoptotic bodies to dendritic cells. Therefore, the induction of apoptosis by replicase-based nucleic acid vaccines not only represents a safety feature, but also seems to be critical for the activation of antigen presenting cells (Leitner et al. 2003). Others have shown that in alphaviral and other vectors, the coexpression of pro-apoptotic factors in DNA vaccines enhances the innate immune response (Chattergoon et al. 2000; Sasaki et al. 2001). Alternatively, Kim et al. (2004) postulate that delivery of DNA by gene gun into the abdominal region of mice directly targets antigen presenting cells. In that case, an enhancement of the immune response using SFV DNA-based vaccines was only possible when the antigen was fused to an antiapoptotic protein (Bcl-xL, Bcl-2, X-linked inhibitor of apoptosis (XIAP)), which enhanced the lifespan of dendritic cells and therefore the duration of antigen presentation to immature T cells (Kim et al. 2004).
It seems that for successful and efficient maturation of dendritic cells, a good balance between optimal production of antigen and apoptotic signals is required. It would be very interesting to test the immunogenicity of a DNA-based equivalent of the SFV(PD) less cytopathic vector.
Cancer therapy. The inherent properties of SFV and SIN vectors to cause a strong induction of apoptosis in the host cell and to highly express exogenous proteins render them very attractive tools for cancer gene therapy. In a tumour therapeutic approach, regression by direct intratumoural injections of SFV has been demonstrated. For instance, two studies reported that administered SFV particles expressing interleukin-12 (IL-12) resulted in significant tumour regression (Asselin-Paturel et al. 1999; Colmenero et al. 2002). In their study, Colmenero et al. (2002) compared the antitumour therapeutic efficacy of antigen-specific and cytokine-mediated recombinant SFV treatments. The results showed that both treatments induced a similar delay in tumour progression. Mice with complete tumour regression developed a long-term immunity. The same group also showed that immunization with tumour antigen-specific recombinant SFV protected mice against subsequent tumour challenge (Colmenero et al. 1999). Several other therapeutic genes were successful as a cancer vaccine when introduced in the SFV vector (Daemen et al. 2002; Yamanaka et al. 2002, 2003). Recently, Tseng et al. (2004) showed that the systemic delivery of SIN–luciferase vectors specifically targets primary and metastatic tumour cells, inducing tumour suppression and eradication. They proposed that specific targeting was achieved due to the inherent property of tumour cells to express excess, unoccupied high-affinity laminin receptors on their surface (Tseng et al. 2004). Specific targeting of SFV vectors has not yet been demonstrated but would be a prerequisite for a clinical application. In an attempt to target tumour cells, several approaches have been undertaken. For example, liposome-encapsulated SFV–IL-12 is being tested in a phase I/II clinical trial (Ren et al. 2003). Certainly, the identification of tumour cell-specific receptors will help the design of new vector strategies. In contrast to its broad host range capacity in vitro, SFV seems to be tissue specific in vivo, depending on the administration route. For cancer therapy, local administration is favoured over the systemic route, because the transduction efficiency seems proportional to the local virus concentration.
Conclusion
So far, SFV and SIN have proven to be very useful vectors for gene delivery into both dividing and postmitotic cells. Investigations are being conducted to determine the critical properties of the vector systems, such as tropism, length and level of transgene expression, immunological responses and toxicity. Modulation of the cytotoxicity by mutating the genome of different viral strains has broadened the spectra of their applications. Further knowledge on the molecular mechanisms activated in host cells upon viral infection will help to adjust SFV and SIN vectors for a wider variety of experimental and therapeutic applications.
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Acknowledgements
The authors thank Dr Sondra Schlesinger for helpful comments and general suggestions on the manuscript, and Dr Christoph Borner for support.
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