Merkel Cell Polyomavirus (MCPyV) in the Context of Immunosuppression: Genetic Analysis of Noncoding Control Region (NCCR) Variability among a HIV-1-Positive Population

Background: Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population. Methods: Urine, plasma, and rectal swabs specimens from a cohort of 66 HIV-1-positive patients were collected and subjected to quantitative real-time polymerase chain reaction (qPCR) for MCPyV DNA detection. MCPyV-positive samples were amplified by nested PCR targeting the NCCR, and NCCRs alignment was carried out to evaluate the occurrence of mutations and to identify putative binding sites for cellular factors. Results: MCPyV DNA was detected in 10/66 urine, in 7/66 plasma, and in 23/66 rectal samples, with a median value of 5 × 102 copies/mL, 1.5 × 102 copies/mL, and 2.3 × 103 copies/mL, respectively. NCCR sequence analysis revealed a high degree of homology with the MCC350 reference strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. In these latter samples, representative GTT and GTTGA insertions were also observed. Search for putative binding sites of cellular transcription factors showed that in several strains, deletions, insertions, or single base substitutions altered the NCCR canonical configuration. Conclusions: Sequencing analysis revealed the presence of numerous mutations in the NCCR, including insertions and deletions. Whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. qPCR measured on average a low viral load in the specimens analyzed, with the exception of those with the GTTGA insertion.


Introduction
Human polyomaviruses (HPyVs) include ubiquitous, clinically silent viral pathogens that establish a symbiotic relationship with their human hosts [1]. Merkel cell polyomavirus (MCPyV) is a small, nonenveloped, double-stranded DNA virus, identified in 2008 [2] and related to the progenitors BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV). Most MCPyV infections are asymptomatic, and serological studies showed that 50-80% of the healthy individuals present MCPyV-specific antibody responses [3,4]. After primary infection, HPyVs establish a lifelong persistence in different anatomical sites, such as lymphoid tissue (JCPyV), renal epithelium (JCPyV and BKPyV), and skin (MCPyV) [5]. Recently, a high prevalence of MCPyV has also been found in respiratory and stool samples from immunocompromised patients, adding important information on MCPyV prevalence and persistence in the respiratory and gastrointestinal tract [4][5][6][7][8]. MCPyV shares the genomic structure of BKPyV and JCPyV, with a circular genome divided into three functional regions: early, late, and interposed between these regions, the noncoding control region (NCCR). The early region encodes the large T antigen (LT), small T antigen (sT), 57 kT antigen (57 kT), and ALTO (alternate frame of the large T open reading frame). The late region is transcribed in the opposite direction and encodes the structural components of the virus capsid: virus protein 1 (VP1) and the minor capsid proteins virus protein 2 (VP2). MCPyV does not seem to express virus protein 3 (VP3), and virions do not seem to contain VP3, despite an in-frame internal adenin-timin-guanine (ATG) start codon in the VP2 gene [9]. The NCCR contains the origin of replication and bidirectional promoter elements [4,10,11]. The two main spliced products, LT and sT antigens, are involved in the viral infectious cycle, in cell cycle progression, and in malignant transformation of the host cell [12]. The continued expression of the MCPyV viral oncogenes sT and LT with a C-terminal truncation is required for Merkel cell carcinoma (MCC) development [13]. Although these findings provide strong evidence that MCPyV is a major causative agent of this skin cancer, whether the NCCR can influence the outcome of the infection remains elusive. It is well documented that one of the remarkable features of NCCRs is the occurrence of rearrangements, which allow to classify BKPyV and JCPyV in archetype (ww) and rearranged (rr) [14][15][16][17]. The archetype form is predominant in the urine of healthy individuals and represents the transmissible form of the virus circulating in the human population and the persistent form in the host. The rr-sequences, characterized by deletions and/or duplications compared with the archetype, are thought to derive from the archetype form during or after reactivation from persistence in vivo, often associated with disease [16,17]. Mutations and rr-forms also emerge in permissive cells in vitro [14,[18][19][20][21], when replication occurs without the constraints of an effective host cellular immune response. The study of the NCCR during host immunosuppression, a critical cofactor for BKPyV, JCPyV, and MCPyV pathogenesis, is important because of the association of the rr-variants with specific human diseases such as polyomavirus nephropathy (PVAN) associated to BKPyV reactivation or progressive multifocal leukoencephalopathy (PML) caused by JCPyV [22][23][24]. Limited data are currently available about the possible relationship between MCPyV NCCRs mutations/rearrangements and the development of MCPyV-related disease. Hashida and coworkers evaluated the genetic variability of MCPyV NCCR in skin swab specimens of healthy individuals with distinct ethnicities and geographic origins, showing that insertions and deletions could be used to classify NCCR into five genotypes. Based on this classification, a tandem repeat was found exclusively in the NCCR of Japanese patients with MCC, while white patients from Europe or North America presented other genotypes [13]. Delbue and colleagues performed the MCPyV NCCR molecular characterization on cerebrospinal fluid (CSF) samples collected from patients affected by neurological disorders. The results obtained showed the presence of MCPyV NCCR IIc strain, according to Hashida's NCCR classification [10]. Thus, Viruses 2020, 12, 507 3 of 15 given this background, the objective of the present study was to investigate the following: first, the prevalence of MCPyV in urine, plasma, and rectal swabs specimens among a HIV-1-positive population; and second, since little is known about NCCR alterations in MCPyV strains circulating in this population, the features of the MCPyV NCCR, focusing on NCCR variability. A search for putative binding sites of cellular transcription factors was also performed in order to verify whether mutations and/or rearrangements detected within the NCCR could fall in these binding sites. The assessment of NCCR region in different anatomical sites could help in advancing the understanding of MCPyV biology and the role of NCCR variability in the context of immunosuppression.

Study Population
A cross-sectional study including a cohort of 66 HIV-1-positive patients, admitted to the Infectious Diseases Clinic of the Polyclinic Tor Vergata Foundation from January 2019 to December 2019, was performed. Among the enrolled patients, 22 were new diagnoses naive to treatment and 44 were experienced patients on treatment with a triple-based antiretroviral regimen including protease/reverse transcriptase/integrase inhibitors. From this cohort (55 males/11 females, age ranged from 21 to 76 years old: mean age ± standard deviation: 40.5 years old; median: 39.9 years old), a sample of urine, plasma, and rectal swab were collected. In detail, 66 plasma, 66 urine, and 66 rectal swabs were obtained for a total of 198 specimens. Demographic and clinical characteristics are presented in Table 1. The study was approved by the local Ethic Committee of the University Hospital Tor Vergata (Rome, Italy) (protocol number 0027234/2018, 19 December 2018), and informed consent was obtained from patients.

MCPyV DNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total DNA was extracted from urine and plasma by DNeasy ® Blood & Tissue Kit (QIAGEN, S.p.A, Milan, Italy) and from rectal swab, using the Stool DNA Isolation Kit (NORGEN BIOTEK, Thorold, ON, Canada) according to the manufacturer's instructions. The extracted nucleic acids were eluted in a final volume of 100 µL, and DNA was evaluated for its PCR suitability by amplifying the β-globin gene sequences [25]. Specific quantitative qPCR assays were performed using TaqMan-based qPCR, employing primers and probes for MCPyV sT, as previously described [26,27]. All samples were tested in triplicate, and the number of viral copies was calculated from standard curves constructed using a ten-fold dilution series of plasmids pMCV-R17a containing the entire genome of MCPyV (Addgene, #24729) (dilution range: 10 8 -10 copies/mL). The lower detection limit of the assay was 10 DNA copies of the target gene per amplification reaction, corresponding to 10 copies per reaction (10 copies/reaction). The results were reported as copies/mL.

MCPyV Nested PCR
MCPyV-positive DNA samples were subjected to nested PCR for the amplification of NCCR region. Two sets of primers, ORIF1/ORIR1 (nucleotide positions 4832-4853 and nucleotide positions 5334-5314) and ORIF2/ORIR2 (nucleotide positions 5077-5100 and nucleotide positions 5280-5261), were employed to generate an NCCR fragment of 504 and 203 base pair (bp). Numbering of nucleotides Viruses 2020, 12, 507 4 of 15 was based on the sequence of MCC350, a strain of North American origin (GenBank: EU375803) [2]. In detail, PCR reactions were carried out following a published protocol [13]. PCR products were analyzed on 2% agarose gels by ethidium bromide staining. The positive PCR products were purified using the MinElute PCR Purification Kit (QIAGEN, Italy) and confirmed by sequencing, using sense and antisense primers, by a dedicated facility (Bio-Fab research s.r.l., Rome, Italy). Sequences were generated using the Big Dye Terminator Sequencing method (Life Technologies) on the ABI 3730 sequencer (Life Technologies, Monza, Italy), and analyzed with the Sequencing Analysis 5.2 software (Life Technologies).

NCCR Alignment and Analysis of Putative Binding Sites
The obtained sequences were compared to the reference strain (EU375803). Sequence alignment was performed using ClustalW2 [28] available on the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) website using default parameters. The identification of putative binding sites for several transcription factors was carried out using TFBIND, available at http://tfbind. hgc.jp/ [29].

Statistical Analysis
MCPyV detection was summarized by counts and proportions. If continuous variables were normally distributed, they were expressed as mean ± SD; if not, they were expressed by median and range. The χ 2 test was performed to evaluate differences for categorical variables. Group differences for continuous variables were tested using Student's t-test or Mann-Whitney U-test, for normally and non-normally distributed variables, respectively. Associations between two continuous variables were examined by Pearson or Spearman correlation for normal or non-normal variables. Differences in viral loads, among the three anatomical sites, were analyzed using Kruskal-Wallis test. A p-value less than 0.05 was considered statistically significant.

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucl 5077 to 5280, were compared with the reference sequence of the prototype North A MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotid 5077 to 5280, were compared with the reference sequence of the prototype North Ameri

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, pla HIV-1-positive subjects was carried out. The amplified NCCRs, spanning 5077 to 5280, were compared with the reference sequence of the prototyp MCC350 [2] ( Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from 5077 to 5280, were compared with the reference sequence of the prototype No

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swa HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide pos 5077 to 5280, were compared with the reference sequence of the prototype North American s MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swa HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide pos 5077 to 5280, were compared with the reference sequence of the prototype North American s MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).  The area highlighted in light grey represents overlapping areas within one or more binding sites. Numbering of nucleotides was based on the sequence MCC350, a strain of North American origin (GenBank: EU375803) [2].
The The area highlighted in light grey represents overlapping areas within one or more binding sites. Numbering of nucleotides was based on the sequence MCC350, a strain of North American origin (GenBank: EU375803) [2].

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine HIV-1-positive subjects was carried out. The amplified NCCRs, spann 5077 to 5280, were compared with the reference sequence of the proto MCC350 [2] (Figure 1).  In the positive plasma samples (7/66, 11%) (MCPyV 26,35,42 belonging to experienced (E) patients and MCPyV 13,21,36,41 belonging to naïve (N) patients), several mutations/deletions were found (Figure 3). In detail, the 5101 T to G transversion, the 5102 G to T transversion, the 5109 T to C transition, the 5148 T to C transition, and the 5220 T to C transition were found in MCPyV 13 ( Figure 3 (Figure 3). In detail, the 5101 T to G transversion, the 5102 G to T transversion, the 5109 T to C transition, the 5148 T to C transition, and the 5220 T to C transition were found in MCPyV 13 ( Figure 3) [2] and that obtained from the sequencing of plasma positive for MCPyV NCCR ( MCPyV 13,21,26,35,36,41,42).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).

Genetic Analysis of NCCR
Analysis of MCPyV NCCR regions obtained from positive urine, plasma, and rectal swabs of HIV-1-positive subjects was carried out. The amplified NCCRs, spanning from nucleotide position 5077 to 5280, were compared with the reference sequence of the prototype North American strain MCC350 [2] (Figure 1).
The main NCCR modifications observed in MCPyV-positive patients, relative to analyzed samples, are summarized in Table 3.

Discussion
A direct link between immunosuppression and the development of opportunistic infections has been established, and it is also known that HPyVs, that commonly infects healthy humans, have a clearly established potential for causing severe organ damage or malignant transformation, especially in individuals with weakened immunity [31][32][33][34]. Although it is well defined that HIV/AIDS predisposes to viral infection and to development of MCC, up to now, very few studies focused on MCPyV prevalence and viral load in HIV-1-positive individuals without MCC [35]. Published data demonstrated that levels of anti-MCPyV IgG in HIV/AIDS patients were significantly higher than those in non-AIDS HIV-infected patients and the prevalence of MCPyV-DNA in peripheral blood mononuclear cells (PBMCs) of HIV/AIDS and non-AIDS HIV-infected patients were 17% and 16%, respectively [36]. Moreover, Fukumoto and colleagues found that 9/23 (39%) serum samples from HIV patients, without highly active antiretroviral therapy (HAART) therapy, were MCPyV-positive [37]. Lastly, MCPyV DNA was found in 2/19 (11%) urine samples of HIV patients, as reported by Torres and colleagues [38].
In this framework, the aim of our study was to evaluate the prevalence and MCPyV loads in urine, plasma, and rectal swabs, among an HIV-1-positive population, in order to better understand MCPyV tissue tropism and to provide new insights on the possible pathogenic role of this virus in human diseases. Our results showed that MCPyV DNA was detected in all type of analyzed specimens, suggesting that the renal epithelium, blood, and gastrointestinal tract could be considered a target of the virus infection. The viral DNA detection confirms that MCPyV is widespread among the population, supporting the urine-oral and fecal-oral routes of transmission [39,40]. In our patients, higher MCPyV prevalence and a higher amount of MCPyV DNA were found in rectal swabs compared with urine and plasma samples. Since some authors reported evidence of HPyVs infection in anal/rectal samples from men who had sex with men, both in HIV-1-positive and in negative population, with higher frequency of MCPyV infection, it is possible to suggest also the sexual route as a possible route of transmission of HPyVs in humans [5,40,41].
Moreover, as suggested by Vergori and colleagues, the higher MCPyV prevalence in rectal swabs could be related to differences in local mucosal immunity activity. In other sites of infection, viral reactivation could be less likely when compared with anal/rectal compartments, where traumatisms and the occurrence of sexually transmitted diseases may decrease the ability of the host to clear virus [5,42]. It is also plausible that the renal epithelium and blood may represent latent sites of the virus, rather than a site of active replication. This assumption is in agreement with HPyVs biology: it is well recognized that primary HPyVs infection is followed by the establishment of an asymptomatic latency state, possibly in the lymphoid, neuronal, kidney, hematopoietic tissues, characterized by low-level replication and excretion, for example, in urine [10,43]. Although no significant association was found between the presence of MCPyV DNA and HIV patient's status at enrollment, MCPyV DNA was found in a higher percentage in naïve patients compared with experienced ones, either in single or all anatomical sites. This data suggest that a weakened immune system with low CD4+ counts as often reported in naïve patients might favor MCPyV replication. In order to improve the knowledge of NCCR alterations in MCPyV strains circulating in the context of immunosuppression, the NCCR variability was analyzed. NCCR rearrangements are described as a pivotal event in the onset of HPyVs-related pathology, as demonstrated for JCPyV and BKPyV in which NCCRs not only control gene expression but also serve as main determinants in viral replication, containing the origin of DNA replication and transcription factor binding sites [15,44]. Instead, little is known regarding the role of NCCR in MCPyV infection, and limited data are available about the relationship between MCPyV NCCR strains and MCPyV pathogenesis. NCCR sequence analysis revealed a high degree of homology with MCC350 strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. Differently to JCPyV and BKPyV, in which the early proximal side of NCCR is highly conserved and the late proximal side undergoes rearrangements [15], we found that insertions and deletions occurred both in early and in late proximal side of the MCPyV NCCR. Specifically, in the analyzed strains, representative GTTGA and GTT insertions (nucleotide positions 5210-5211) were observed in both plasma and rectal swabs. Interestingly, these alterations were found exclusively in male gender with a statistical difference very close to significance (p = 0.054). The MCPyV NCCR structure, in contrast to JCPyV and BKPyV, has been previously associated with the geographic origin of the patients [13]. Hashida and colleagues identified two major subtypes of MCPyV NCCR, subtypes I and II, with the presence or absence of a 25 bp tandem repeat (TGTCCTCCTCCCTTTGTAAGAGAAA) into nucleotide positions 5177-5178, respectively. Based on the occurrences of two additional insertions (2 bp, TT, and 5 bp insertions, GTTGA, between nucleotide positions 5199-5200 and 5210-5211, respectively), MCPyV strains were assigned further to five genotypes. In our analyzed strains, we found the MCPyV NCCR IIa-2 strain in plasma and rectal swabs, which contain the 5 bp insertion and represents the predominant strain among white persons of European descent, as predictable for our cohort of patients [13]. In this study, we evaluated beside the NCCR rearrangements the possible nucleotide changes that fell within the putative binding sites for cellular transcription factors [13,30,44,45]. Sequence analysis showed that MCC350 NCCR sequence contains the NF1, NFκB, Tst-1, OCT1, AP-1, and TATA binding sites already described within the NCCRs of other HPyVs [13,30,44,45]. In several strains obtained from MCPyV-positive samples, deletions, insertions, or single base substitutions fell within these putative binding sites. It can be envisaged that some of these changes did not allow the identification of putative binding motifs such as Sp1 and/or p53, already described in the NCCR of other HPyVs [13], in our NCCR sequences. The relevance of cellular factors such as Tst-1, NF-1, Sp1, NF-kB, and PURα, that specifically determine JCPyV tropism for glial cells and play an important role in efficient HPyVs DNA replication, is well described [46]. Moreover, a potential association, between a C/G mutation in the NCCR Sp1 site and increased BKPyV virulence in hemorrhagic cystitis (HC) patients, has been proposed [47]. In light of these findings, further studies are warranted in order to define the importance of these NCCR binding sites and understand how their changes (mutations, insertions, or deletions) may drive MCPyV replication and subsequent in vivo pathogenicity. Finally, in the attempt to understand whether rearrangements could be correlated to a higher replicative capacity, the median MCPyV viral load of each district was analyzed in relation to the presence of rr-NCCRs, confirming that the MCPyV strains that carried the GTTGA insertion in NCCR showed a higher rate of replication compared with the ones without insertion, either in the same or different anatomical sites. As previously reported for BKPyV and JCPyV, rr-NCCRs conferred a higher replication rate to these viruses contributing to diseases progression [22]. Consequently, also for MCPyV, it is possible to speculate that the shift from canonical NCCR to rr-NCCR could determine higher replication capacity and increase the pathogenic potential in the context of immunosuppression. It is also likely that high MCPyV-DNA levels might increase the chance of its integration into the host cell genome and, then, its oncogenic properties in a context different from that of MCC.

Conclusions
Based on the high prevalence of MCPyV in rectal swabs, we suggest that sexual route can represent another route of transmission of HPyVs among humans. Although sequencing analysis revealed the presence of numerous mutations in the NCCR, whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. The study adds important information about MCPyV prevalence, viral load, and NCCR behavior in HIV-positive individuals and suggests to monitor the possible role of MCPyV in triggering disease outside the MCC context. Author Contributions: Conceptualization, C.P., M.C., and V.P.; Formal Analysis, A.N. and C.d.V.; Resources, L.C., L.S. and M.A.; Investigation, C.P., F.O., D.A., S.P., P.C. and D.M.R.; Data Curation, C.P., M.C., and V.P.; Writing-Original Draft Preparation, C.P., M.C., and V.P.; Writing-Review & Editing, C.P., P.C., M.C., V.P., L.S., M.A., A.T.P.; Supervision, V.P. and A.T.P.; Funding Acquisition, V.P. and A.T.P. All authors have approved the submitted version and agree to be personally accountable for the author's own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work are answered. All authors have read and agreed to the published version of the manuscript.