- Research article
- Open Access
Gypsy endogenous retrovirus maintains potential infectivity in several species of Drosophilids
BMC Evolutionary Biologyvolume 8, Article number: 302 (2008)
Sequences homologous to the gypsy retroelement from Drosophila melanogaster are widely distributed among drosophilids. The structure of gypsy includes an open reading frame resembling the retroviral gene env, which is responsible for the infectious properties of retroviruses.
In this study we report molecular and phylogeny analysis of the complete env gene from ten species of the obscura group of the genus Drosophila and one species from the genus Scaptomyza.
The results indicate that in most cases env sequences could produce a functional Env protein and therefore maintain the infectious capability of gypsy in these species.
Gypsy (gypsyDm) is an endogenous retrovirus of D. melanogaster. Its structure is largely similar to the vertebrate retroviruses and it possesses infective properties [1–4]. Sequences homologous to gypsyDm are widely distributed among Drosophila species, found in both the subgenus Sophophora as well as in the subgenus Drosophila [5–9]. In addition, they have been detected in some species of the genus Scaptomyza [8, 9]. The phylogenetic relationships among gypsy sequences from Drosophila species is not be always coincident with those of their hosts, which could be indicative of horizontal transfer during evolutionary history [6, 7, 9, 10]. Horizontal transfer has been invoked to explain the evolutionary patterns of several families of transposable elements [11–15]. In some cases, such as the P element, the horizontal transfer events are strongly documented [16–18]. However, the underlying mechanisms to horizontal transfer remain elusive.
In the case of gypsyDm, the horizontal transfer could be associated with the infectious properties of that element. One way to evaluate the potential impact of the gypsy infectious ability on the evolutionary history of gypsy sequences from Drosophila, is to analyze the infective ability of gypsy homologous sequences from species other than D. melanogaster.
The infectious ability of gypsyDm is associated with the expression of a retroviral envelope-like protein encoded by the env gene . Structural analysis of the env region of a given gypsy sequence is the first step in determining its potential infectious ability. In previous reports, two complete gypsy elements from D. subosbcura and D. virilis were sequenced [10, 19]. Although, the env region is preserved in its full-length, in both species single indels result in potentially truncated Env proteins . However, as has been proposed in , it is possible that both, defective and complete fully functional gypsy elements, coexist in the genome of these species. This study describes the existence of full-length env genes in the genomes of D. subosbcura and D. virilis, as well as in the genomes of the several species closely related to D. melanogaster:D. simulans, D. erecta, D. orena, D. teissieri, and D. yakuba. Interestingly, these gyspy sequences can potentially invade the D. melanogaster genome, and escape the control normally exerted by the flamenco gene, whose product represses mobility.
It is generally accepted that species of the obscura group can be classified into five subgroups: the pseudobscura and affinis subgroups, consisting of Neartic species, the subobscura and obscura subgroups, mainly consisting of Paleartic species, and the microlabis subgroup encompassing Afrotropical species [20–22]. Gypsy sequences homologous to gypsyDm have been detected in several species of the Neartic and Paleartic subgroups, appearing as a monophyletic group that is highly diverged from the gypsyDm prototypic element [7, 8]. With the exception of D. guanche, the 5' region of the env gene in all species examined could potentially produce a functional N-terminal region of the Env protein. To confirm the existence of potentially infective gypsy lineages other than gypsyDm, gypsyDs, and gypsyDv, we report here an analysis of the complete env gene of ten species of the obscura group and one species from the genus Scaptomyza. Most of the species analyzed contain intact copies of the env region and the env splicing sites, and in vitro can be translated into a protein of the predicted size.
Results and discussion
Sequences homologous to gypsy from D. melanogaster are widely distributed among Drosophila species. In previous studies, the complete gypsy elements from D. subobscura and D. virilis have been sequenced . These studies were carried out to understand the evolutionary behaviour of gypsy and indicate that the genetic organization of gypsy in those species is conserved. Moreover, in D. melanogaster do the gypsy elements have a complete functional env gene . In the case of D. virilis and D. subobscura, it has been shown that the genomes of these species contain at least one copy of gypsy putatively encoding a complete envelope protein .
The main aim of this study is to examine whether potentially functional copies of the env region of gypsy exist in other species. This was accomplished by searching genomes for the presence of full-length env coding sequences and assessing their functionality.
The analysis of the envsequences shows their infectious capability
To analyze the infectious capability of the gypsy element of each analyzed species (table 1) we amplified the env region with specific primers designed from the intergenic sequence (figure 1). In all species the size of the amplified sequence was 1.5 kb, except for D. algonquin and D. tolteca, with sizes of 0.5 and 1.2 kb respectively. The PCR products of all species, were cloned in pCR®2.1-TOPO® (Invitrogen) and sequenced with the specific primers of the vector, demonstrating that D. algonquin and D. tolteca had a deletion of 1.5 kb and 0.3 kb respectively. In the same way, D. pseudoobscura and D. miranda had deletions of 40 pb and 21 pb respectively that produced a stop codon and an Env truncated protein. D. persimilis had a single nucleotide change, also producing a stop codon. In contrast, D. madeirensis, D. obscura, D. ambigua, D. bifasciata, D. affinis and S. elmoi had different polymorphisms but none resulting in a truncated protein, so these env regions would produce potentially active proteins.
With these data, we can conclude that D. madeirensis, D. obscura, D. ambigua, D. bifasciata, D. affinis and S. elmoi could produce a complete Env protein and therefore could maintain the infectious capability of gypsy in these species.
The envsequences obtained in different species have the capacity to produce complete envelope proteins
The RTS system was used to scan for premature termination mutations and analyze the coding capacity of the gypsy env gene present in the genome of the different species studied. We used genomic DNA from each species to amplify the env sequence. We cloned the obtained sequences into pCR®2.1-TOPO (Invitrogen) and then we used these clones to produce the PCR products for in vitro protein expression (see material and methods).
The major protein products obtained in this assay have molecular weights corresponding to the expected size for Env protein of D. melanogaster, about 54 kDa. These protein products were obtained in the same species where the env nucleotide sequence was complete, without disruptions. We obtained complete Env protein from D. madeirensis, D. obscura, D. ambigua, D. bifasciata, D. affinis and S. elmoi, while we obtained truncated proteins from the species where the sequence was disrupted, D. pseudoobscura, D. persimilis and D. miranda (figure 2).
In D. melanogaster, the splicing produces a start codon using AT from 5' site in the gag region and a G from the 3' site in the env region . We have analyzed the sequences from the different species of this study and only D. madeirensis, D. obscura, D. ambigua, D. bifasciata, D. affinis and S. elmoi, have conserved the sequences that produce the start codon after the splicing.
These results show that these species contain at least one copy of gypsy element that can encode an envelope protein. Other data that support the existence of functional Env proteins include the presence of N-glycosylation sites, conserved cysteine residues and endopeptidase cleavage sites in all the species with a complete env sequence and the ATG codon (figure 3), as described in [2, 4]. Moreover, those species have the sequence pattern (R-X(2)-R-X(5,6)- [GE]-X(5)- [LV]-X-G-X(2)-D-X(2)-D) for the detection in silico of endogenous retroviral envelope protein proposed in . Only the signal peptide and the membrane-spanning domain sequence show differences on comparing to D. melanogaster gypsy sequence.
Although sequences have the capability to produce complete envelope protein, their expression could be repressed in the species studied
To demonstrate the capability of the gypsy elements to produce an active envelope protein, we wanted to know if the env gene was expressed in vivo in the species with the complete env sequence.
In order to detect the production of the env mRNA we performed a RT-PCR analysis using the GS05 and GS06 primers with the mRNA extracted from ovarian chambers of the female flies. Since we also wanted to detect alternative splicing, necessary to produce ENV, we used the primers GYPSY5'F and GS06 of the env gene using the same mRNA as before (see materials and methods).
Amplification with the internal primers was successful in all species analysed as well as in the positive control (data not shown). However, alternative splicing was not detected (data not shown). These results suggest that the alternative splicing of gypsy in the obscura species group and S. elmoi would be repressed by a genomic gene as occurs in D. melanogaster with the flamenco gene . Alternatively, the gypsy element may use an alternative start codon to express the env region or we simply could not detect the alternative splicing.
The gypsyphylogeny is not totally consistent with species phylogeny
Since gypsy elements can potentially jump from one individual to another without the need of a vector, it has been proposed that horizontal transfers of gypsy elements is favoured by their ability to encoded full-length ENV proteins . In this way we performed a phylogeny analysis of the env gypsy sequences studied, looking for evidences of horizontal transfer events.
A Neighbor-joining phylogenetic tree of envelope sequences is shown in figure 4. The obscura sequences fall into four main clades that are generally consistent with species phylogeny. Clade A includes sequences from D. affinis of the affinis subgroup; clade B, D. ambigua, D. bifasciata, D. madeirensis, and D. obscura of the obscura subgroup; clade C, D. miranda, D. persimilis, and D. pseudoobscura of the pseudoobscura subgroup; and clade D, D. tolteca of the affinis subgroup. The D. virilis sequence is responsible for the relatively low support for clade A; removing the D. virilis sequence from the analysis increases the bootstrap values to 98/77 for a grouping of the D. hydei sequence with those from D. affinis, with the rest of the topology unchanged. With the exception of the placement of sequences from S. elmoi, the topology of this tree is identical to that obtained by parsimony analysis, which yielded a shortest tree of 1477 steps (Consistency Index = 0.8; Retention Index = 0.9). The distribution of sequence from D. affinis and D. tolteca into two distinct clades is an unexpected result that is consistently seen in all analyses. Constraining the analysis so that the sequences from D. tolteca and D. affinis are monophyletic adds an additional nine steps to the parsimony tree.
The potential effect of recombination on the phylogeny was assessed by using the GARD algorithm, which uses maximum likelihood models to detect recombination events. The BIC model found no evidence of recombination, while the AIC model identifies a potential breakpoint at position 1288 in the 1392 character alignment. The potential effect of this event on the phylogeny was also examined using a sliding-window approach. Separate phylogenetic analyses were constructed from four 400-bp regions of the envelop alignments (positions 1–399; 400–799; 800–1199; and 1200–1493), and on a fifth dataset that excluded the region beyond the breakpoint. The latter analysis resulted in an identical tree to that shown in figure 4, as did two of the four sliding window analyses. In the other two sliding-window trees, the D. virilis sequence was excluded from clade A, as it was in one of two alternate trees obtained from the AIC analyses. Regardless of its precise affiliation, in an expanded phylogenetic analysis that includes 180 published gypsy sequences, D. virilis clearly belongs to a well-supported clade that includes all of the obscura sequences (see below).
Sequences from S. elmoi fall into clade B, in which they form two subclades. Although not monophyletic, the average amount of sequence divergence between the two subclades is just 1.1%, and if the sequences from S. elmoi are constrained to be monophyletic the total length of the parsimony tree is unchanged. If the gypsy sequences from S. elmoi are excluded from the analysis, the topology of the obsucra sequences is unchanged from that shown in figure 4. To make the gypsy phylogeny conform completely to the species phylogeny (including S. elmoi as an outgroup, a monophyletic clade that includes D. hydei and D. virilis, and D. melanogaster as a sister taxon to the obscura sequences) increases the parsimony tree length from 1477 to 1542 steps. Thus, there is clear incongruence between the gypsy phylogeny and that of the species from which the sequences were isolated.
The envelope sequences from S. elmoi differ on average by only 2.1% from those found in the obscura species of clade B. As a hallmark of horizontal transfer is the existence of highly similar sequences in the genomes of divergent taxa , transfer of gypsy from the obscura group to the genus Scaptomyza seems likely. Because the sampling from S. elmoi is quite extensive, an explanation of the retention of an ancestral polymorphism for the phylogenetic incongruence seems unlikely. This conclusion is further supported by comparison to the divergence of a non-mobile nuclear gene, Adh. Over exon 2 of this gene, the average divergence between S. elmoi and the clade B Adh sequences is 20.0%. A horizontal transfer involving the genus Scaptomyza and the Palearctic members of the obscura species group is of particular interest because of the well-documented horizontal transfer of the P transposable element between these taxa . In that case, the direction of transfer was almost certainly from Scaptomyza to D. bifasciata. In the case of gypsy, the phylogeny indicates a transfer in the opposite direction.
To provide some comparison to other studies of gypsy evolution, an extensive phylogenetic analysis was performed with an enlarged dataset of 180 sequences, combining those from this study with those of  and . This allows us to assess the coherence of the two clades, A and B, which include postulated horizontal transfer events, and to increase the sample size considerably to examine the alternative hypothesis of ancient lineages. This tree is presented in figure 5. The positions of the D. hydei, D. virilis and S. elmoi sequences remain unchanged from that shown in figure 4, providing additional evidence to support the hypothesis of horizontal transfer.
Molecular evolutionary analysis of gypsysequences is consistent with activity
To examine the evidence for selection on the gypsy sequences, the numbers of synonymous substitutions (dS) and nonsynonymous substitutions (dN) were calculated for each sequence. The average dN/dS ratio was then calculated for each clade (see table 2), providing some indication of whether purifying selection is or has been operating on these sequences. For all major clades, the dN/dS ratio is similar to that obtained for non-mobile nuclear genes . Similar results have been obtained in a number of studies of gypsy sequence evolution [6, 8–10]. Together, these comparisons suggest that many gypsy elements are capable of activity, consistent with the expression data described above. Although the pattern is consistent with activity (and potential infectivity), this conclusion may be complicated by two factors: (i) codon bias, which is lower for the gypsy sequences, when compared to Adh (data not shown), and (ii) the nature of selection on the gypsy sequences which may include functional constraints on the RNA intermediate in addition to selection for a functional envelop protein.
It is noteworthy that clade B includes the gypsy sequences that have intact envelope genes (D. ambigua, D. bifasciata, D. madeirensis, D. obscura, and S. elmoi), whereas clades D and C consist of those with interrupted reading frames. Clade A comprises sequences from the Neartic species D. affinis, which also encode full-length envelope proteins and are characterized by very low dN/dS ratios. It is fascinating that in the two clades with open envelope reading frames and low dN/dS ratios, horizontal transfer has likely occurred. Even though the gypsy sequences examined from clade C are not likely functional, the low dN/dS ratio suggests that these elements have been active in the recent past. The envelope coding regions of D. tolteca (clade D) are characterized by a substantial deletion at the beginning of the envelope gene and relatively higher dN/dS ratios, suggesting that they have been inactive for considerably longer than the clade C elements. Thus, the phylogenetic and sequence analyses together provide a snapshot of the evolutionary history of gypsy in the obscura species group that is consistent with their activity and potential infectivity.
Horizontal transfer of gypsy has been suggested in other studies: among sequences from the obscura group and D. virilis/D. hydei, which belong to a different subgenus , and within the melanogaster species group . Two additional studies, which are similar is scope to the one described here, concluded that horizontal transfer of gypsy was fairly widespread among species in the genus Drosophila and sister genera [9, 26]. As these studies included a total of only three sequences from the obscura group, they were not able to identify precisely the species involved in the transfer events. In the analysis presented here, the envelope sequence from D. hydei shows a strong affiliation to the sequences from D. affinis; a less well-supported clade includes the sequences from D. virilis as well. As the envelope sequences from D. hydei, D. virilis, and D. affinis differ by an average of only 6.2%, and the species sampling is now quite extensive (see figure 5), horizontal transfer remains the best explanation for the incongruent phylogeny. The hypothesis of horizontal transfer finds additional support in a comparison of the divergence of the envelop region to the non-mobile gene, Adh. The average divergence of Adh sequences from D. hydei/D. virilis and D. affinis is 21.4% (clade A).
Using the values for synonymous nucleotide substitutions,  provided an estimate of 6.3 million years ago (MYA) for a transfer from the obscura group (as represented by D. persimilis) to D. virilis. When a similar analysis is done with the D. affinis sequences (which are not included in ), an estimate of only 2.7 MYA is obtained for a transfer from D. affinis to D. hydei, and 3.1 MYA for a transfer from D. affinis to D. virilis. In clade B, time estimates calculated from synonymous changes place the transfer from the Paleartic obscura to S. elmoi at 2.1 MYA. The fact that these transfers occurred relatively recently supports the idea that at least some genomes in the obscura group harbor active, infectious gypsy elements.
The genomes of six of the eleven species of analysed encode intact copies of the envelop coding region of gypsy and retain intact envelop splice sites. Their potential for expression was analyzed in vitro and each was found to encode a protein whose size is consistent with activity. Moreover, these elements possess two additional features of active gypsy genes, alternative splice sites and the use of alternative start codons. Evolutionary analysis of the envelop sequences shows that interspecies transfer of gypsy is associated with those obscura elements that can encode a functional envelop protein. Such horizontal transfer is supported by the incongruence of the gypsy and species phylogenies and by the low levels of gypsy divergence when compared to the divergence of host genes from the same species.
Drosophila and Scaptomyzastocks
Ten species from the obscura group (D. madeirensis, D. obscura, D. ambigua, D. bifasciata, D. pseudoobscura, D. persimilis, D. miranda, D. affinis, D. algonquin, D. tolteca), and one species from the Scaptomyza genus (S. elmoi) were analysed. All species were obtained from the Tucson Stock Center (table 1).
PCR amplification of the envcoding region
Genomic DNA from each species was prepared as in . PCR was usually carried out using 100 ng of genomic DNA, 10 pmol of each primer, 10 mM dNTPs and 0.5 U of Netenzyme DNA polymerase. The amplifications were performed in a Eppendorf Mastercycler 5333 version 2.01.33-09. The amplified 1443-bp fragment contains a short intergenic stretch, and the entire ORF3 region from the gypsy element. The pairs of primers used to amplify the env region in the differents species were (see figure 1): ENV5'mad: 5'-AGTAGAGTTAGAATAACGTCC-3' and GYPSY3': 5'-TRGCGMGTCAGCATTGTT-3' (where R is G or A) for D. madeirensis, D. ambigua, D. bifasciata and S. elmoi; ENV5'obs: 5'-ATTAGAGTTAGAACAACGT CT-3' and GYPSY3' (see sequence above) for D. obscura; ENV5'pseuaff: 5'-CTTAGAGTTAGAACACCGTCT-3' and GYPSY3' for D. pseudoobscura, D. persimilis, D. affinis, D. algonquin; ENV5'mir: 5'- CTTAGAGTTAGAACACCAT CT-3' and GYPSY3' for D. miranda; ENV5'tol: 5'- CTTAGAGTTAGAACACCTTCT-3' and GYPSY3' for D. tolteca.
PCR conditions consisted of a single denaturing step at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 55–60°C for 1 min (depending on each primer), and 72°C for 1 min 30 s; a final extension was performed at 72°C for 10 min.
To amplify the 5' region of gypsy, which contains the env splicing site, different primers, derived from alignments of gypsy elements from D. subobscura, D. virilis and D. melanogaster were designed. The size of the amplified fragment is 500 bp. The sequences of these primers were (see figure 1): GYPSY5'F: 5'-GCKWTGATGGCGTATGCATTG-3' (where K is G or T and W is A or T) and GYPSY5'R: 5'-YTATGCTGCCGAAAGTATGC-3' (where Y is C or T).
Cloning and DNA sequencing
PCR products were cloned into pCR®2.1-TOPO® (Invitrogen). Eight clones from each species were sequenced in an automatic ABI-Prism sequencer. The multiple alignments of gypsy ORF3 sequences were performed with the program SEQUENCHER version 4.0.5 (Gene Codes Corporation). The sequencing reactions were carried out with the primers (see figure 1): ENV5' and GYPSY3' (see sequence above) GS05, 5'-TAA TAC TCA CGA TAA CGT TGG-3' for D. bifasciata, D. madeirensis and D. ambigua, GS04, 5'-TAA TAC TCA CCA TAA CGT TGG-3' for D. affinis and GS06, 5'-ATG TCC GAT GAT GTT TAG GAG-3', GS02, 5'-CACTTAAATTCAACTTTGGGG-3' and GS03, 5'-CCAAAGTTGAATTTAAGTGCC-3' for all species.
All sequences obtained in this study were submitted to the EMBL Nucleotide Sequence Database [EMBL: AM748829 – AM748898].
Phylogenetic and sequence analysis
Nucleotide sequences were aligned using the default settings of ClustalW http://www.ebi.ac.uk/clustalw/ and were then refined by eye to correct obvious misalignments. The phylogeny of 64 gypsy sequences from the obsura group and seven sequences from Scaptomyza elmoi was examined using parsimony, distance, and maximum likelihood methods as implemented by PAUP* 4.0b10. Four additional gypsy sequences, from D. subobscura [X72390], D. hydei [X74539], D. virilis [M38438], and D. melanogaster [Z31368] were also included in the analysis. A total of 1392 positions were analysed, with gaps treated as missing data. Identical tree topologies were obtained for all major clades using each method, and adjusting the search parameters for each. The Neighbor-Joining tree shown in figure 4 was constructed using the Kimura two-parameter model for nucleotide substitution. The parsimony analyses, which included 553 informative characters, used random addition of sequences (10 replicates) and TBR branch swapping; both heuristic and branch-and-bound searches yielded identical trees. The maximum-likelihood analyses used empirical base frequencies and a substitution model in which all rates were equal.
Previous studies of gypsy phylogeny indicate that the gypsy sequence from D. melanogaster is an appropriate outgroup for phyolgenetic analysis of sequences from the obscura group (e.g., ). The use of this taxon as an outgroup was verified in two additional ways. First, it is clearly the most divergent of the gypsy sequences, showing an average distance of 36% (Kimura two-parameter method) from the other sequences. Second, midpoint rooting with both neighbor-joining and parsimony establish the root along the D. melanogaster branch. Bootstrap values were determined from 1000 replicates for both parsimony and Neighbor-Joining. Character state analyses and parsimony tree-length comparisons were determined with MacClade 4.0.3. The potential effect of recombination on the phylogenetic analyses was assessed with the GARD method .
Molecular evolutionary analysis was implemented with MEGA4 . Nucleotide divergences were calculated using the Kimura two-parameter method and the numbers of synonymous substitutions (dS) and nonsynonymous substitutions (dN) were calculated using the Nei and Gojobori (1986)  codon model for the nucleotide sequences that had been aligned based on amino acid position. To facilitate comparisons, the alignments were adjusted by eye to maintain homologous codon positions. Because of some variability in identifying the start site in all sequences, the comparison began at a conserved serine codon, which, in most sequences, is the eighth amino acid of the envelope polypeptide. The divergence of gypsy sequences was compared to those of a non-mobile nuclear gene, Adh. Sequences for D. hydei, D. virilis, S. elmoi, and all available taxa of the obscura species group were obtained from GenBank. The comparisons were limited to exon 2, the only sequence available for some species.
In vitro translation of the Env proteins
To generate PCR products for in vitro protein expression, special primers, consisting of two different parts, were designed. The first half adds regulatory elements necessary for expression in a prokaryotic system, such as a T7 promoter, the ribosomal binding site and the T7 terminator. The second half recognizes a specific region of env, which corresponds to the sequence used to amplify the env region in the different species (see sequences above). This PCR product can be expressed directly in E. coli HY using the Rapid Translation System (RTS) from Roche. The E. coli Linear Template Generation Set, His-tag and the RTS 100 E. coli HY (both purchased from Roche), were used to detect mutations that lead to the termination of mRNA translation and subsequently to protein truncation. The translation products were separated by discontinuous SDS-PAGE through a 12% separating gel with Tris-glycine buffer. The signals were detected by colourimetric system.
RNA extractions were carried out using approximately 100 mg of flies. These RNA extractions were carried out with the QuickPrep™ Micro mRNA Purification Kit (Amersham Biosciences), according to the manufacturer's instructions. Afterwards, the mRNA was treated with RQ1 RNase-free DNase (Promega) to remove remainder DNA of the sample. RNA obtained was reverse transcribed in the presence of AMV-RT (Promega) and the env-specific primer GS06 (see sequences above).
Reactions were carried out at 37°C for 2 hours followed by 10 minute at 65°C to denature the enzyme. From the first strand reaction, 2 μl was used as a template in PCR using the following primers: GS05, for D. bifasciata, D. madeirensis and D. ambigua, GS04, for D. affinis and GS06 (see sequences above) for all species (figure 1).
To analyse the alternative splicing product, the RNA obtained was reverse transcribed and amplified with GS05 and the primer GS06.
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This work was supported by a grant (BMC2000-1446) from the Spanish Ministerio de Ciencia y Tecnología (I+D).
JVLL carried out the molecular genetic studies, the obtention of the sequences, participated in the sequence alignment and analysis and drafted the manuscript. JBC carried out the phylogenetic and sequence analysis and drafted the manuscript. IMG participated in the sequence alignment. SS participated in the submission of the sequences to the EMBL Nucleotide Sequence Database and the draft the manuscript. RF participated in the conception and the design of the study. MJMS conceived and designed the study and coordinated and helped to draft the manuscript. All authors read and approved the final manuscript.