RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques. RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. It has been known since 1949 . TherefoThe aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the high-throughput selective 2′-hydroxyl acylation analyzed via the primer extension (hSHAPE) methodology, discussing examples taken from the world of viruses and how they have influenced the probing techniques since 2005. Indeed, nowadays, we have numerous sophisticated tools to assess RNA structure in vitro, in cellula, and even in viro, either alone or in complex with various macromolecules. These technological breakthroughs using enzymatic and chemical probes, which are detected through advanced methods , were maSince the first mention of RNA, referred to as “yeast nucleic acid” in the early 1900s, numerous key discoveries have been made concerning its structure. In 1954, the hypothesis that RNA is a “2′-3′-5′ branched” molecule was abandoned in favor of the linear 5′-3′ chain . In 1956Aspergillus oryzae by Sato and Egami ethane, also known as mustard gas . This coFrom 1965 to 2005, 40 years of RNA probing research has led to the development of a wide range of tools available to scientists .Neurospora crassa endonuclease  5′-end radiolabeled RNA. Since the discovery of these self-cleaving RNAs, they have been widely used as tools for controlling gene expression [Many viral RNA structures are known to carry out important functions during replication. These functions often require the viral or/and cellular partners recruited by structural RNA motifs. However, some of the structures present in viruses, viroids, and virusoids are functional on their own once they have adopted a specific 3D structure . This is IVS RNA . In this IVS RNA , and the IVS RNA , and [32pression .2-nucleotides, this laboratory had noticed that the 2′ position of nucleotides with a flexible sugar ring, which generally correspond to unpaired nucleotides, was more reactive than that of base-paired nucleotides [Asp [Since the beginning of chemical probing, probes have been selected for their ability to highlight the exposure to the solvent, flexibility, or ss nature of the nucleotides. A combination of a few probes was usually required, in order to establish an experimentally validated secondary structure model of an RNA of interest. This changed in 2005, with the work of Merino et al. . Indeed,leotides . Therefodes [Asp . The res1/2 = 33 min and 73 min, respectively) [The first notable improvement brought by SHAPE was that it allowed for the interrogation of the 4 nucleotides of RNA in a single experiment and, thus, to map all the reactivities of an RNA molecule using a single probe ,107,108.ctively) ,113.Another important improvement concerns the coupling of SHAPE mapping with adduct detection by capillary electrophoresis (CE), in order to allow for high-throughput and quantitative analysis (hSHAPE). Indeed, the initial approach consisted of extending radioactive primers and visualizing the cDNAs obtained by PAGE, but the methodology quickly evolved with the use of fluorescently labeled primers. In that case, the cDNAs obtained after the reverse transcription step can be separated by capillary electrophoresis and quantified on DNA sequencers  Figure .The switch from PAGE to capillary electrophoresis has had multiple consequences. First of all, the reproducibility and resolution of the results have greatly improved, which has allowed for obtaining information for every nucleotide and to increase the length of the reading windows from approximatively 80–100 nucleotides to 300–400 nucleotides in a single experiment, making the study of large RNAs more accessible . Second,SHAPE was successfully applied to the study of the RNA structures of several human pathogens. Using 1M7, Watts et al.  were ablBesides the hSHAPE technique, other “old probes” have also been coupled to CE detection, in order to gain more information on RNA structures. Actually, SHAPE probes interrogate the sugar face of the RNA, but information on the Watson–Crick face accessibility are also of interest; chemical probes, such as DMS ,130,131 Even if hSHAPE and its further modifications constituted important advances in RNA structure determination by decreasing the workload of the experiments and facilitating quantification, the improved, but still limited, output and the large amount of RNA required for the detection of the modified nucleotides were the two biggest limitations of this technique. Indeed, for studies that are limited to small regions (and when the amount of RNA available is low), it is preferable to use the older method, consisting of the extension of radiolabeled primers, followed by analysis by PAGE, instead of the extension of fluorescent primers analyzed on a DNA sequencer. The coupling of RNA probing with next-generation sequencing (NGS) allowed the community to overcome these limitations and step into a new era of transcriptome- and genome-wide studies of RNA structure (structurome).Structurome studies began in 2010 with enzymatic probes, and two methods were developed almost simultaneously: parallel analysis of RNA structure (PARS)  and fragN-cyclohexyl N′-(2-morpholinoethyl) carbodiimide, the reactive group of CMCT, was also used in the Seq technique to map the pseudouridine positions in rRNA [Given the advances made possible by SHAPE-seq, “old” probes have also been revisited, in combination with deep-sequencing detection. A multitude of techniques have emerged that often vary only in one of the steps of the library preparation. HRF-Seq  takes ad in rRNA , as well in rRNA ,146  structures are important in the virus life cycle ,149, andMoreover, other improvements have been made to the “PROBE-Seq” techniques by introducing a second and orthogonal reactive group inside pre-existing probes. Using bi-functional probes, such as N-propanone isatoic anhydride (NPIA) in the SHAPES technique , NAI-N3 Mutational profiling (MaP) was perhaps the most important improvement in determining the structure of RNAs by simplifying and reducing biases during library preparation. It was first adapted to SHAPE as SHAPE-MaP ,158 and Recently, new chemical probes were designed and adapted to the MaP strategy . Under lNevertheless, the probing techniques described above are still limited because, for example, they do not directly identify base-pairing interactions, rather they infer them from the compatibility that may exist between the observed reactivity and the proposed structure. That is why even more sophisticated methods have been developed. Among them, the “RNA interaction groups analyzed by mutational profiling” (RING-MaP) technique  allows fAnother interesting application of MaP is the localization of the protein interaction sites on RNAs by the RNP-MaP technique . Indeed,There is no doubt that these latest developments have greatly improved the study of RNA structures and will pave the way for more in-depth studies. However, performing such experiments, involving a high rate of RNA chemical modifications, may induce structural changes during the probing experiment, and careful controls must be carried out to avoid analysis bias.These powerful techniques were applied to the study of several viruses. In recent years, the global structure of the influenza A virus (IAV) genome was undertaken by SHAPE-MaP experiments, with a 1M7 probe in vitro and inside virions . The autSince 2020, the highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has also been studied with mutational profiling approaches. SHAPE-MaP ,178 and Research on enzymatic and chemical probes in the second half of the last century has led, in barely 20 years, to the creation of a toolbox of probes that complement each other and that have allowed for the identification of many RNAs structures, as well as ligand-binding sites. Quickly after their discovery, these techniques have successfully been applied to the study of viral RNAs and have enabled a better comprehension of the biological roles of their structural features. However, the notable shortcomings of these methods were their inability to quickly provide robust structural information, as the generation of data was slow and quantification of the experiments was difficult, complicating data analysis.In 2005, these shortcomings were addressed with the development of a new class of chemical probes targeting the sugar 2′-OH, regardless of the nature of the base and, later, the use of fluorescent primers and analysis of the cDNAs by capillary electrophoresis. These advances gave a new impetus to the RNA structural probing field by allowing for large-scale analyses in a shorter time, with quantifiable results that were easier to reproduce and compare.Most importantly, these steps in the direction of high-throughput probing have paved the way for even more performant techniques: the next-generation sequencing-based RNA structure probing technologies. The methods initially developed with enzymatic probes were abandoned over time, in favor of the more versatile chemical probes. Indeed, one after another, almost all the former chemical probes have been coupled to deep-sequencing, which provides a more diverse and adaptable array of tools, available for the study of many RNA species and contexts.As the novel combinations of probes with various methods based on NGS technology become more common, new challenges will arise in the RNA structural probing field. Thanks to the decades of development of the processing algorithms, the RNA secondary structures can be predicted in silico with or without probing information, even if experimental data are required to sharpen and confirm the predicted models. This points toward an increasing need for tools, in order to address the 3D structures of RNA molecules, as many functions and mechanisms rely on long-range interactions and complex folding structures. Some steps have already been taken toward this goal, with the recent development of SHAPE-JuMP , but alsBesides, a new window is presently opening in the field of RNA probing with nanopore sequencing, which allows for the direct detection of the RNA modification on individual (even very long) molecules, without the need for a reverse transcription step. The coupling of nanopore sequencing with RNA sequencing should prove especially useful in the analysis of co-existing RNA structures, which is difficult with ensemble techniques .Finally, it is also crucial to remember that the structural information in the RNA field is required to decipher the functions carried out by RNA molecules. In order to establish a clear link between RNA motifs and their molecular functions, new ultrahigh-throughput methods were developed, which showed promising results in predicting the impact on single elements of RNA functions .


Coronavirus (CoV) genomes consist of positive-sense single-stranded RNA and are among the largest viral RNAs known to date (∼30 kb). As a result, CoVs deploy sophisticated mechanisms to replicate these extraordinarily large genomes as well as to transcribe subgenomic messenger RNAs. Since 2003, with the emergence of three highly pathogenic CoVs , significant progress has been made in the molecular characterization of the viral proteins and key mechanisms involved in CoV RNA genome replication. For example, to allow for the maintenance and integrity of their large RNA genomes, CoVs have acquired RNA proofreading 3′-5′ exoribonuclease activity . In order to replicate the large genome, the viral-RNA–dependent RNA polymerase  is supplemented by a processivity factor , making it the fastest known RdRp. Lastly, a viral structural protein, the nucleocapsid (N) protein, which is primarily involved in genome encapsidation, is required for efficient viral replication and transcription. Therefore, CoVs are a paradox among positive-strand RNA viruses in the sense that they use both a processivity factor and have proofreading activity reminiscent of DNA organisms in addition to structural proteins that mediate efficient RNA synthesis, commonly used by negative-strand RNA viruses. In this review, we present a historical perspective of these unsuspected discoveries and detail the current knowledge on the core replicative machinery deployed by CoVs. Indeed, in contrast to DNA organisms, RNA-dependent RNA polymerases (RdRps) that ensure viral RNA genome synthesis are devoid of coreplicative and postreplicative fidelity-enhancing pathways . TheAmong eukaryote RNA viruses, those with positive polarity genomes (+RNA) can be directly used by host ribosomes to produce viral proteins. By convention, they are divided into structural proteins and proteins for viral genome amplification ). For the latter, in addition to the key RdRp activity already mentioned, some other essential enzymatic activities are commonly found in +RNA viruses, such as protease and RNA helicase. Besides, depending on the virus family, less common or unique activities can be found. In any case, cytoplasmic viral nsps assemble in protein complexes, which, in association with modified intracellular membranes, form viral replication organelles. They offer a favorable microenvironment for the amplification of the viral genome through a full-length minus-strand intermediate, serving in turn as a template for new viral messenger RNA genome production. In addition, these viral organelles may shield viral dsRNA intermediates from innate immune sensors. Neosynthesized +RNA genomes can then either be translated into additional viral proteins, serve as a template for additional minus-strand RNA synthesis, or be packaged into progeny virions.Coronaviridae family, which together with the Arteriviridae, Roniviridae, and Mesoniviridae families belongs to the Nidovirales order  were mainly a veterinary problem with substantial economic losses , whereas endemic human CoV strains (such HCoV-229E and OC43) were considered benign to human health. They typically caused mild upper respiratory tract diseases or the common cold. It was only at the beginning of the 21st century that CoVs became a real threat to mankind, with the emergence of severe acute respiratory syndrome-CoV (SARS-CoV) in December 2002 , 6, and es order . In addihe order , 11, 12.L. nidus = nest). sg mRNAs are expressed from a complex mechanism involving discontinuous RNA synthesis. Remarkably, these sg mRNAs harbor common 5′- and 3′-terminal sequences with the full-length genome.Nidoviruses are positive-sense single-stranded RNA viruses, comprising the largest viral RNAs known to date (from 12.7 to 41.1 kb) . The emeIn this review, we relay the history of the discoveries and detail the current knowledge on the core replicative machinery deployed by CoVs to replicate, transcribe, and maintain the integrity of their extraordinarily large genome (∼30 kb for the three highly human pathogenic CoVs). Whenever possible, a parallel is made with arteriviruses, the short vertebrate nidoviruses that share the same genomic organization as well as sg mRNAs transcription, but without ExoN proofreading activity.The polycistronic RNA genome of CoVs is flanked by two untranslated regions (UTR) at its 5′ and 3′ ends. The 5′UTR, of about 350 nucleotides (nt), contains a 5′ cap structure followed by the leader and a transcriptional regulatory sequence (TRS), both involved in sg mRNA production (discussed later). The CoV 3′UTR consists of 300 to 500 nucleotides plus a poly(A) tail of approximately ∼47 nt on average . Both geNidovirales frameshift event frequency at the ORF1a/1b junction is between 15% and 70%, the accepted efficiency for CoVs is around 50%  essential for viral replication  that are directly accessible to cellular ribosomes . This leound 50% , 18, 19.lication . The finectivity , 21.Figupro), also known as 3CLpro (due to its homology to the 3Cpro of picornavirus), is established (pro in nsp5)  . Thus, vin nsp5) . The fulin nsp5) . While tin nsp5) . They alThe 16 released functional CoV nsps, with a multitude of host proteins, form protein complexes and as such are referred to replication–transcription complexes (RTCs) , 27. Remversus the different sg mRNAs (transcription). Indeed, as will be illustrated in this review, the CoV replicative cycle is fine-tuned by the dynamic associations and dissociations of, at least, some small ORF1a-encoded proteins (nsp7 to nsp10)  . Other no nsp10) .As with all eukaryote +RNA viruses, these viral replicase protein complexes are localized in specific organelles that are viral-induced from host cell membranes. There is a dual benefit for +RNA viruses, by concentrating all the elements for RNA genome replication as well as hiding viral negative-strand RNA and dsRNA intermediates from innate immunity receptor detection . For Coe.g., modulation of the cellular innate immune response) , envelope (E), membrane (M), and nucleocapsid (N) proteins, as well some species-specific accessory proteins that are not essential for virus replication but are involved in pathogenesis (esponse) . Indeed,esponse) . Notablyesponse) . Thus, iesponse) , 32, 40.The different sg mRNAs are generated through a unique discontinuous transcription mechanism controlled by body-TRS. All TRSs contain a conserved 6 to 7 nt core sequence surrounded by variable sequences. Thus, a body-TRS is found just upstream of each ORF that encodes structural and accessory proteins, as well at the 5′ end of the genome  . The preOverall, the viral RTCs recognize different RNA promoter sequences and produce many viral RNAs. Indeed, CoVs produce the (+) RNA genome, a variable set of sg mRNAs (eight for SARS-CoV), and negative-sense RNA intermediates, being at least 20 different RNAs. In addition, numerous other viral RNAs coming from noncanonical discontinuous transcription events have been detected in the case of SARS-CoV-2  as well As outlined earlier, CoVs employ a unique, sophisticated multi-subunit machinery in order to replicate and maintain the integrity of their genome as well as transcribe a set of sg mRNAs encoding their structural and accessory proteins. Since 2010, the understanding of this machinery at the molecular level has significantly progressed. Most of the functional and structural information of the replicase machinery is available for SARS-CoV, MERS-CoV, and SARS-CoV-2, which we describe in the following sections.in vitro nsp12-RdRp activity was obtained . Thereby, as putative cofactor(s), SARS-CoV nsp12 protein partners, previously identified by different methods  in the presence of nsp8 and nsp7 was discovered (via a flexible peptide linker between both), suggesting a rate-limiting association between both. Further analyses showed that the nsp7–nsp8 complex activates and confers processivity to the nsp12-RdRp  activities), hence retaining all associated enzymatic activities (in vitro proofreading system reconstitution (discussed later). The first demonstration of nsp12-RdRp processivity factor requirements was performed for SARS-CoV (A). An nsp7-nsp8 heterodimer binds to the thumb, and an additional copy of nsp8 binds to the finger domain. In light of these structural data, the nsp7-nsp8 heterodimer plays a critical role in stabilizing the polymerase domain, thereby enabling template recognition and binding (A) (While the first CoVs that infect humans (HCoV-229E and HCoV-OC43) were isolated in the 1960s, it was only in 2014 that a highly active CoV RNA polymerase has been identified . Indeed,obtained , 48. Thi methods , 50, 51, methods . Followiscovered . Then, op12-RdRp . Indeed,p12-RdRp . Importativities . This prSARS-CoV  but has SARS-CoV  and SARSSARS-CoV . MoreoveSARS-CoV  and thenSARS-CoV , 65, 66.SARS-CoV A. An nsp binding . Moreove binding A 57). T. Tin vitIn contrast and surprisingly, biochemical RNA polymerase activity for arteriviruses, the short nidoviruses, remains elusive, and this is despite attempts to supplement arterivirus RdRp assays with the corresponding region of pp1a as a potential cofactor by analogy of the distantly related CoV RdRp cofactors .via two distinct mechanisms: either de novo or, in contrast, via a short RNA primer. For the latter, viruses have evolved different priming mechanisms, such as the formation of a covalent complex between the RNA primer and a viral protein  or cap-za virus ). In theza virus , but theza virus . Thus, mIn line with their exceptional genome size, CoV RNA-synthesizing machinery is the fastest known RdRp but also the least accurate , 73. Thivia complementary enzymatic activities (see below). More broadly, all nidoviruses harbor this unique amino-terminal domain extension adjacent to the RdRp domain (Arteriviridae family. Self-nucleoside monophosphatylation of EAV nsp9 (ortholog of CoV nsp12) has been reported in vitro, with UTP and GTP as preferred substrates. Consequently, it was named NiRAN for nidovirus RdRp-associated nucleotidyltransferase domain . They contain a very small number of conserved residues among all monophyletic viral families but highly diverged Nidovirales order. Moreover, these conserved residues are essential for EAV in addition to SARS-CoV replication in cell culture  harboring the RdRp domain in CoVs is much larger than that of other (+) RNA viruses, with a size of ∼106 kDa against ∼65 kDa, traditionally. This feature comes from a unique amino-terminal extension that is inextricably linked to viral RNA polymerase activity and supports the latter p domain . From a e domain . This nu culture . Further culture . In addi culture . In this culture . The ide culture . Neverthaviridae . Indeed,aviridae .Moreover, the NiRAN domain appears to possess more than one function. Indeed, very recently it has been shown that SARS-CoV-2 nsp12 NiRAN possesses guanylyltransferase (GTase) activity, the first step for the formation of the cap structure at the 5′ end of viral mRNAs . Universde novo initiating RNA polymerase, able to synthesis products of less than six nucleotides   . In contoV-229E) . Lastly,oV-229E) . To conci.e., eight molecules of nsp7 with eight molecules of nsp8), forming a ring with a central channel that through its size and electrostatic properties can perfectly accommodate a dsRNA  tail  tail . Whether(A) tail , 66, 96.(A) tail . Notably(A) tail , which t(A) tail . Among tin vitro nsp14-ExoN enzymatic activity was demonstrated   . One majviruses)  and thenviruses) . With thlication , 102. Lalication . This stlication , TGEV . Through this pathway, the antiviral compound ribavirin 5′-monophosphate was significantly incorporated by the SARS-CoV polymerase complex but also readily excised from RNA. This result may explain its limited efficacy on CoV-infected patients , a beta-barrel domain, and a C-terminal superfamily 1 helicase core with two RecA-like subdomains , 121. Thactivity , 127. Inactivity . The samactivity . Neverthin vitro SARS-CoV-2 replicative machinery reconstitutions. Indeed, in these structures, it was mostly revealed two nsp13 molecules sit above the polymerase complex (made up of nsp12-nsp82-nsp7) (B). By analogy to cellular DNA and RNA polymerases, it was proposed that the RNA-bound nsp13 molecule would push the RdRp backward on the template RNA. Indeed, this reversible phenomenon, called backtracking, is well-known and -characterized in eukaryotic and prokaryotic cells  that runs opposite to that of the RNA polymerase (3′→5′) means that direct cooperativity between both, that is, helicase unwinding the RNA template for copying, cannot be considered. A solution to this conundrum comes from recent structural studies of 82-nsp7) , 62. The82-nsp7) B. By anaic cells , 131. Thic cells . The avaic cells . This roic cells . To concin vitro CoV reconstituted replicative system, combined with advances in deep sequencing, CoVs’ transcriptome could be fully characterized and defined. The temporality and spatial arrangement of proofreading during RNA synthesis also remain to be unraveled.Since the emergence of SARS-CoV in 2003, significant progress has been made in the characterization of key mechanisms and proteins involved in the replication of the RNA genome. The emergence of SARS-CoV-2 has literally boosted our knowledge, notably regarding the structural organization of the key viral proteins involved in these processes. Nevertheless, many gray areas remain such as the transcription mechanisms of the sg mRNAs with potential “strand jump”. In the future, with an Nidovirales order.Finally, it is important not to be limited to SARS-CoV-2/pathogen CoVs but to extend our knowledge to the different families of viruses forming this exceptional and fascinating The authors declare that they have no conflicts of interest with the contents of this article.


Each coevolution signal detected was tested and verified by global-fit cophylogenic analysis using software ParaFit, PACo, and eMPRess.Bat-borne viruses are relatively host specific. We hypothesize that this host specificity is due to coevolution of the viruses with their hosts. To test this hypothesis, we investigated the coevolution of coronavirus and paramyxovirus with their bat hosts. Published nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) gene of 60 coronavirus strains identified from 37 bat species, the RNA polymerase large (L) gene of 36 paramyxovirus strains from 29 bat species, and the cytochrome B (Significant coevolution signals were detected in coronaviruses and paramyxoviruses and their bat hosts, and closely related bat hosts were found to carry closely related viruses.Our results suggest that paramyxovirus and coronavirus coevolve with their hosts. Filoviridae , Paramyxoviridae , and Coronaviridae  [Bats are reservoirs of many zoonotic viruses, such as members of virus-2) , 2. Batsvirus-2) .Coronavirinae under the family Coronaviridae . Bats coronaviruses have been shown to be responsible for the outbreaks of severe acute respiratory syndrome (SARS) in 2002–2003, Middle East respiratory syndrome (MERS) in 2012 [Coronaviruses are taxonomically placed in the subfamily  in 2012 , 5, and  in 2012 .Henipavirus of the family Paramyxoviridae  and have been detected in flying fox bats (Pteropus spp.) [Henipavirus causes severe symptoms associated with high mortality rates in humans and livestocks. HeV was first detected in Queensland, Australia in 1994, causing acute respiratory disease and febrile illness in horses and humans who have close contact with sick horses [Some paramyxoviruses such as Hendra virus (HeV) and Nipah virus (NiV) are highly pathogenic zoonoses. Both viruses belong to the genus us spp.) , 8. Henik horses –12.cytB) gene of 61 bat species with those of the RNA dependent RNA polymerase (RdRp) gene of 60 coronavirus strains and the RNA polymerase large (L) gene of 36 paramyxovirus strains.Both coronaviruses and paramyxoviruses have a certain degree of host specificity. Host–parasite specificity has also been observed in malaria parasites , bat fliRhinolophus affinis found in Yunnan Province, China is on the same branch of the phylogenetic tree as the 18 human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains examined, suggesting that it is a relative of SARS-CoV-2 . When each individual sequence was analyzed by the Individual host–parasite (H–P) link test function of ParaFit, 51 of the 60 bat coronavirus strains were found to have a significant coevolution relationship (link) with their bat hosts with a ParaFit1 or ParaFit2 P value ≤ 0.05.Analyses of all bat Miniopterus bats. Strains SAX2011/CHN/KJ473806, Anlong-57/CHN/KY770851, and Anlong-43/CHN/KY770850 are also related and are all from Myotis bats. Similarly, KW2E-F151/GHA/KT253269 and AT1A-F1/GHA/KT253272 are related and are all from Hipposideros bats. These observations suggest host specificity of these bat coroanviruses. The following coroanviruses are closely related but are from different species of bats of the same family (Vespertilionidae): Lushi-212/CHN/KF294373 (from Murina leucogaster), 16715 23/VIE/MH687934 (from Scotophilus kuhlii), HKU6-1/CHN/DQ249224 (from Myotis ricketti), and CDPHE15/USA/KF430219 (from Myotis lucifugus)  and JTMC15/KU182964 (99.68% RdRp sequence identity) found in the same species (Rhinolophus ferrumequinum) of bats in Jilin Province, China [Aselliscus stoliczkanus (family Hipposideridae) that are evolutionarily close to Rhinolophidae bats  bats that are distantly related to Rhinolophidae bats cytB gene sequences and all paramyxovirus RNA polymerase large (L) gene sequences as a whole by the Global test function of ParaFit and PACo showed evidence of coevolution between bat paramyxoviruses and their bat hosts . When each individual sequence was analyzed by the Individual H–P link test function of ParaFit, 7 of the 36 bat paramyxovirus strains were found to have a significant host–parasite coevolution relationship (link) with a ParaFit1 or Parafit2 P value ≤ 0.05.Analyses of all bat Hipposideros bats (family: Hipposideridae) , and the hosts of the other four strains are bats of the Pteropodidae family, including Desmodus rotundus, Carollia perspicillata, Carollia brevicauda, and Glossophaga soricina . Therefore, we, concluded that these host–virus pairs have coevolved Fig. .Fig. 4P-In this study, we investigated the coevolution relationship between bats and their viral parasites: coronaviruses and paramyxoviruses. These two groups of viruses were chosen because they have been shown to be zoonotic . The seqMiniopterus pusillus, Miniopterus fuliginosus, and Miniopterus magnater that are very close to each other. Another example is the coevolution relationship between closely related bats including Tylonycteris pachypus, Hypsugo savii, Vespertilio sinensis, Neoromicia capensis, and Ia io and the following coronavirus strains: GX2012/CHN/KJ473822, 206645-40/ITA/MG596802, SC2013/CHN/KJ473821, NL13845/CHN/MG021451, 5038/RSA/MF593268, and PML-PHE1/RSA/KC869678. Similar coevolution relationships are found in closely related bat species Rhinolophus ferrumequinum, Rhinolophus blasii, Rhinolophus pusillus, Rhinolophus macrotis, Rhinolophus sinicus, Rhinolophus pearsonii, and Rhinolophus affinis and the following coronavirus strains: RF1/CHN/DQ412042, JTMC15/CHN/KU182964, JL2012/CHN/KJ473811, 16BO133/ROK/KY938558, HeB2013/CHN/KJ473812, SX2013/CHN/KJ473813, Shaanxi2011/CHN/JX993987, HuB2013/CHK/KJ473814, HKU3-1/CHN/DQ022305, YN2013/CHK/KJ473816, Rs3367/CHN/KC881006, GX2013/CHN/KJ473815, Anlong-103/CHN/KY770858, Rp3/CHN/DQ071615, LYRa11/CHN/KF569996, and RaTG13/CHN/MN996532  of coronavirus strains but only 19% (7/36) of paramyxovirus strains have a significant coevolution relationship with their bat hosts. Since significant coevolution was found in both groups of the viruses by eMPRess and ParaFit and PACo Global tests, this low positive individual H–P link rate in paramyxoviruses may be due to small sample size.Hypsugo savii) and 206645-63/IT/MG596803 (bat host: Pipistrellus kuhlii) share 99.46% nucleotide sequence identity in their RdRp gene and are found in bats living in the same geographical area, Italy. In addition, coronavirus strains 16BO133/ROK/KY938558, JL2012/KJ473811, and JTMC15/KU182964 are highly related with > 99.6% nucleotide sequence identity in the RdRp gene and are found in bats distributing in areas near each other, including Jilin Province, China (for isolates JL2012/KJ473811 and JTMC15/KU182964) and South Korea (for isolate 16BO133/ROK/KY938558) that is close to China.We postulate that that evolutionary relationship and close habitat of bat species contribute to inter-species transmission of viruses. One observation supporting this hypothesis is that coronavirus strains 206645-40/IT/MG596802  [Myotis daubentoniid, Eidolon helvum, Hipposideros abae, Hipposideros ruber, and Hipposideros pomona were found to harbor both coronavirus and paramyxovirus. Altogether, 61 different species of bats were identified to be the hosts of the coronavirus and paramyxovirus strains examined in this study. As the cytochrome B (cytB) gene is one of the most conserved gene in bats, its sequence was used for coevolution analyses. The cytB sequences of 59 of the 61 bat species were downloaded from the GenBank. Since the cytB gene sequences of Miniopterus pusillus and Tylonycteris robustula, that were the hosts of coronavirus strains 1B/CHN/EU420137 and HKU33/CHN/MK720944, respectively, were not available, they were determined in this study. These two bat species were captured from Menghai, Yunnan and Kau O Bat Cave, Macau, respectively. Anal swabs of Miniopterus pusillus and a small portion of the patagiums of Tylonycteris robustula were obtained. The captured bats were released back to their roosts after sampling. DNA was isolated from these samples and used as the template for amplication by polymerase chain reaction (PCR) of a portion (1140 bp) of the cytB gene with primers L14727ag (5′-ATGATATGAAAAACCATCGTTG) and H15915ag (5′-TTTCCNTTTCTGGTTTACAAGAC) [The database of Bat-associated Viruses   containsACAAGAC) . PCR conhttps://www.ebi.ac.uk/Tools/msa/clustalo) [Megaderma lyra/DQ888678. Maximum-likelihood phylogenetic trees were constructed using 1000 bootstraps with the raxmlGUI program [To analyze the nucleotide sequences, they were aligned with Clustal Omega  . The out program  using th program .To determine the degree of congruence between bat and virus topologies on phylogenetic trees and identify individual associations contributing to the cophylogenetic relationship the software ParaFit was used . ParaFitTo obtain comparable global goodness-of-fit statistics with Parafit global values, the software package Procrustean Approach to Cophylogeny (PACo)  in R wasThe event-based program eMPRess  was used


Small non-coding RNAs (sncRNAs) represent an important class of regulatory RNAs involved in the regulation of transcription, RNA splicing or translation. Among these sncRNAs, small nucleolar RNAs (snoRNAs) mostly originate from intron splicing in humans and are central to posttranscriptional regulation of gene expression. However, the characterization of the complete repertoire of sncRNAs in a given cellular context and the functional annotation of the human transcriptome are far from complete. Here, we report the large-scale identification of sncRNAs in the size range of 50 to 200 nucleotides without a priori on their biogenesis, structure and genomic origin in the context of normal human muscle cells. We provided a complete set of experimental validation of novel candidate snoRNAs by evaluating the prerequisites for their biogenesis and functionality, leading to their validation as genuine snoRNAs. Interestingly, we also found intergenic snoRNAs, which we showed are in fact integrated into candidate introns of unannotated transcripts or degraded by the Nonsense Mediated Decay pathway. Hence, intergenic snoRNAs represent a new type of landmark for the identification of new transcripts that have gone undetected because of low abundance or degradation after the release of the snoRNA. The continuous progress in the annotation of mammalian genomes first led to the striking conclusion that they are mostly non-coding, with only 2% having information to encode proteins . In addiWe have also reported that a single transcription unit could in fact generate multiple RNA species, including long or short coding, or non-coding RNAs depending on alternative splicing (AS) events of introns, whereby introns can be either spliced or retained in the host transcript ,6,7,8. HBeing removed from the pre-mRNA to allow for the formation of mature mRNA, the intron lariat is then disconnected by the Debranching enzyme 1 (DBR1) and rapidly degraded within minutes after its excision. Intrinsically non-coding and with a short half-life, a subset of introns nevertheless shows high sequence conservation across closely related species, suggesting functional constraints on intronic sequences throughout evolution . Many stWe previously referred to regulatory ncRNAs originating from intron splicing as Short Intron-Derived small ncRNAs (SIDs) . SIDs inNumerous studies have expanded the repertoire of the human transcriptome with new small ncRNAs ,33,34,35+) RNAs to enrich sncRNAs independently of their host genes while eliminating abundant mature miRNAs. We applied this strategy to the context of human muscle progenitor cells and uncovered around 400 yet unannotated sncRNAs. Biocomputational predictions revealed that one-third corresponded to snoRNAs, 14% corresponded to new pre-miRNAs and 8% corresponded to snRNAs. Since snoRNAs were assumed to be all annotated, although recent studies have pointed out that it was not quite the case [In order to capture the full repertoire of small ncRNAs without a priori knowledge of their biogenesis, structure and genomic origin, we designed a “medium” RNA-seq on total RNAs with a size range of 50–200 nt, compatible with the known size of sncRNAs precursors, and depleted in rRNAs and poly-depleted RNAs extracted from human myoblasts (MBs) and their differentiated myotube (MT) counterparts. The RNAs were size-fractionated to isolate the fraction > 50 and <200 nt in length and further sequenced using the medium RNA-seq procedure described in the Material and Methods section and in For the rest of the study, we decided to focus on the 175 predicted new snoRNAs. As shown in In order to classify the newly identified snoRNA candidates as genuine snoRNAs, they have to satisfy several prerequisites: (i) transcription and splicing of their host gene, (ii) incorporation into snoRNP complexes and (iii) accumulation in nucleoli.TNPO2 showed no correlation (r = 0.04) and the pair #186/DDX60L even showed anti-correlated expression levels (r = −0.99). As a whole, the mean correlation coefficient of expression levels of the 68 snoRNA candidates and their corresponding host genes was 0.31. These data indicate that the expression levels of the snoRNA candidates do not reflect that of their host genes, suggesting the existence of posttranscriptional mechanisms.Since most canonical human snoRNAs are processed from the splicing of introns , snoRNA We randomly selected 21 unannotated intronic sncRNAs predicted as snoRNA candidates to experimentally validate them as genuine snoRNAs . Among tTo test whether the snoRNA candidates were incorporated into snoRNP complexes, we performed immunoprecipitation (IP) of the Dyskerin, Fibrillarin and Coilin proteins from nuclear extracts of muscle cells, followed by RT-PCR to detect the snoRNA candidates . WesternIn sum, almost all of the snoRNA candidates that we tested were found associated with snoRNP or scaRNP complexes and could therefore represent new snoRNAs with genuine functions of snoRNAs.Since canonical snoRNAs are located in nucleoli, in part to guide the modification of rRNAs , we asseAltogether, these results established that most of the snoRNA candidates tested were associated with snoRNPs and accumulated in nucleoli, suggesting that they can be considered as new genuine snoRNAs.From all of the small ncRNAs identified by the medium RNA-seq, 137 were considered intergenic since they were not embedded in known and yet annotated genes in hg19 or hg38 builds. Amongst these 137 intergenic sncRNAs, 49 were identified as snoRNAs using blast and dedicated software (see above). This observation raised the question of how these snoRNAs were transcribed since they were not embedded within a gene body. As above, we randomly selected 16 newly identified intergenic snoRNAs to assess their incorporation in snoRNPs and their localization in nucleoli. Among the 16 tested, 12 new intergenic snoRNAs can be considered genuine snoRNAs A,B.ATP11C gene in the hg38 annotation. To assess if the levels of intergenic snoRNAs were dependent on splicing machinery, i.e., to test whether other intergenic snoRNAs could uncover introns of yet unannotated transcripts, we performed shRNA-mediated RNA interference against the general splicing factors U1 small nuclear ribonucleoprotein 70 kDa (SNRNP70), SNRNP40 (PRP8) and Splicing factor U2AF 65 kDa subunit (U2AF65)  . As show(U2AF65)  used as (U2AF65) C, sugges(U2AF65) C. This iWe then took advantage of the total RNA-seq to investigate whether other intergenic snoRNAs could be hosted within introns of yet unannotated transcripts. We reconstructed transcriptional units (TUs) surrounding intergenic snoRNA candidates using STAR and Scallop as accurate reference-based transcript assemblers that feature high accuracy in assembling multi-exon transcripts as well as weakly expressed transcripts. From the results obtained, we reliably identified TUs and splicing junctions surrounding the candidates #293, #342, #351 and #366 . As an experimental validation, we performed RT-PCR on MBs to detect the new TUs. However, we could only detect TU#342 from total RNA fractions (data not shown), suggesting that the candidate TUs might belong to the fraction of weakly expressed non-coding transcripts that preferentially locate in specific subcellular compartments. We therefore performed RT-PCR on RNAs isolated from cytoplasmic or nuclear extracts. Surprisingly, we detected PCR products of TU#293, TU#351 and TU#366 in nuclear but not in cytoplasmic fractions, consistent with our hypothesis A. TU#342Altogether, these findings suggest that the majority of newly identified intergenic snoRNAs can be reclassified as genuine snoRNAs . In addiHere, we identified and experimentally validated new snoRNAs in the context of human muscle progenitors, which contributes to continuous improvement of the annotation of the human genome. Interestingly, half of the new snoRNAs were found in intergenic regions, which is quite remarkable since, at least in mammals, most snoRNAs are processed from the splicing of introns. We provided evidence that these intergenic snoRNAs are indeed hallmarks of the presence of not yet annotated transcription units. Previous studies aimed to systematically identify new small ncRNAs from yeast to human through plants, either on the basis of bioinformatics predictions to search for specific structures or motifs, or via specific sequencing methods dedicated to the identification of ncRNAs in a given size range (reviewed in ). For exWe identified yet unannotated snoRNAs in the context of human muscle progenitors, either unique or belonging to already known families of snoRNAs but with distinct genomic locations. To fully experimentally validate these snoRNA candidates as genuine snoRNAs, we reasoned that they must fulfill certain requirements such as transcription of the host gene in the chosen context, their presence in snoRNP complexes and nucleolar localization. The host genes of the snoRNA candidates were indeed transcribed in the muscle context, as seen from the total RNA-seq, although there was no correlation between the levels of expression of the host gene and that of the corresponding snoRNA. This is also consistent with the number and the expression levels of the snoRNAs showing very little variation during muscle differentiation. This absence of correlation was already observed in numerous studies ,52,53. ThnRNP), RNA helicases or proteins involved in splicing rather than with the canonical methylating protein complex in order to regulate alternative splicing  II promoter have already been described, although they do not seem to function as genuine snoRNAs . Rather  the NMD . This ty the NMD ,46. As a whole, we identified new sncRNAs and experimentally validated new unannotated snoRNA genes in the context of muscle cells. In addition, we provide evidence that new intergenic snoRNAs were in fact located within introns of new transcriptional units that can be used as hallmarks for the identification of yet unannotated SNHGs relevant for muscle function.We used myoblasts (MBs) and in vitro differentiated myotube (MT) counterpart cell lines . Cells w®, and subjected to Bioanalyzer. Only RNAs with a RIN > 8 were kept for further libraries construction and sequencing . Sequencing was performed with a 75-base-pair read length in a pair-ended manner.For the total RNA-seq, RNAs were isolated as described below using TRI Reagent+) RNAs using three rounds of NEBNext Poly(A+) mRNA magnetic isolation module  according to the manufacturer’s instructions except that supernatants containing poly(A) depleted RNAs were recovered by phenol/chloroform extraction. Then, RNAs were depleted in rRNAs using the Ribo-Zero™ Magnetic Gold Kit . RNAs wider than 200 nt were removed using NucleoSpin miRNA kit  and RNAs smaller than 50 nt were removed by gel purification using the methods described by Ellington and Pollard [For the medium RNA-seq, RNAs were first depleted in poly. Alignments were performed using Star  for the total RNA-seq and Bowtie2 for medium RNA-seq using hg19 from UCSC. The genomic data generated for this study were deposited in the GEO repository with the accession number GSE178649.The primary antibodies used were directed against Dyskerin , Fibrillarin , Coilin , Lamin A/C , α-Tubulin , Nucleophosmin , small nuclear Ribonucleoprotein 70 , U2AF65 , snRNP40  and irrelevant antibody .®  according to the manufacturer’s instructions and as previously described [® miRNA  according to instructions [Total RNA was isolated using Tri reagentescribed ,7. Shortructions .RNA was isolated as described above and reverse transcribed as described previously . PrimersThe cells were lysed, and the RNA and proteins were extracted and quantified as described previously ,7 and us2, Roche’s complete Protease Inhibitor Cocktail and Vanadyl ribonucleoside complexes ), layered over 3 mL of Solution II  and centrifuged at 1430× g for 5 min at 4 °C. Then, the nuclei pellet was resuspended in 3 mL of Solution II and sonicated in 10 s ON/10 s OFF six times in cold water using Diagenode Bioruptor UCD-200 at power setting “High”. The sonicated nuclei were checked under a phase contrast microscope to ensure that more than 90% of the nuclei were broken. Next, the sonicated solution was layered over 3 mL of Solution III (0.88 M sucrose and 0.5 mM MgCl2) and centrifuged at 2800× g. The supernatant corresponding to the nucleoplasmic fraction was removed and stored at −80 °C. The pellet containing the nucleoli was washed twice by resuspension in 500 µL of Solution II and centrifuged at 2000× g for 2 min at 4 °C. Finally, isolated nucleoli were resuspended in 500 µL of Solution II, transferred into a new tube and stored at −80 °C until further use. Total, cytoplasmic, nuclear, nucleoplasmic and nucleolar proteins were analyzed by SDS–PAGE and immunoblotting as previously described [Nucleoli isolation was performed as described previously  with minescribed , or whenescribed .Purified nucleoli were spotted on poly-L-Lysine slides  and air dried. The slides were rehydrated with PBS for 5 min at room temperature (RT) and incubated with anti-NPM1 antibody (1:100) for 30 min at RT. Then, the slides were washed three times with PBS and incubated with goat anti-mouse IgG  for 30 min at RT. Finally, the slides were washed three times with PBS, counterstained with 0.66 mM Pyronin Y  for 1 min, washed again three times with PBS and mounted with Vectashield.Short hairpin RNAs (shRNA) directed against human snRNP70, U2AF65, PRP8 and Luciferase were produced using MessageMuter™ shRNA production kit  and in vitro transcribed using T7 RiboMax large-scale production system (Promega) following manufacturer’s instructions. Sequences are depicted in The knockdown of snRNP70 + U2AF65 was combined with that of the major splicing factor PRP8, since interference of snRNP70 or U2AF65 alone was insufficient to reduce splicing significantly in another study . shRNAs The myoblasts (MBs) were seeded the day prior the treatment to reach 70% confluency. Then, the MBs were treated with actinomycin D to inhibit the transcription by the RNA polymerase II (1 µg/mL). Total RNA was extracted at 0 h, 1 h, 4 h, 24 h and 48 h after the addition of the drug. Finally, the RNA levels of the candidates were assessed by RT-PCR, as described above.The myoblasts (MBs) were seeded at 70% confluency. Twenty-four hours later, the culture medium was replaced with a medium containing caffeine (10 mM) for 8 h. The total RNA from untreated and treated MBs was isolated as described above.


Non-coding RNAs (ncRNAs) comprise a set of abundant and functionally diverse RNA molecules. Since the discovery of the first ncRNA in the 1960s, ncRNAs have been shown to be involved in nearly all steps of the central dogma of molecular biology. In recent years, the pace of discovery of novel ncRNAs and their cellular roles has been greatly accelerated by high-throughput sequencing. Advances in sequencing technology, library preparation protocols as well as computational biology helped to greatly expand our knowledge of which ncRNAs exist throughout the kingdoms of life. Moreover, RNA sequencing revealed crucial roles of many ncRNAs in human health and disease. In this review, we discuss the most recent methodological advancements in the rapidly evolving field of high-throughput sequencing and how it has greatly expanded our understanding of ncRNA biology across a large number of different organisms. Escherichia coli in 1984 [Caenorhabditis elegans [The central dogma of molecular biology that Francis Crick proposed in 1957 described a model for the flow of information between genes and proteins . Additio in 1984 , and Xis in 1984 , and mic elegans , in the In parallel to the discoveries of diverse RNAs and their roles in the field of molecular biology, a novel technology for the determination of the sequences of nucleic acids appeared on the horizon. The first DNA sequencing approach has been developed by using a chain termination approach established by Sanger and colleagues in 1977 . Based oThe latest major advance in sequencing technology is the development of long-read sequencing . Long-read sequencing is largely brought about by two novel technologies: Single-molecule real-time (SMRT) sequencing and nanopore sequencing, which are commercialized by Pacific Biosciences and Oxford Nanopore respectively . SMRT seThe development of different sequencing techniques enables faster and more cost-efficient sequencing and improved quality of sequencing data . AdditioThis review summarizes the recent development in ncRNA sequencing and describes the impact sequencing technology has had on the field of ncRNA biology. We focus on how RNA sequencing has been an important tool to uncover novel ncRNAs  as well While genome sequencing efforts came to fruition upon the turn of the century, one major question which remained open was: How much of the genome is transcribed? In order to address this question, the RIKEN institute decided to start a consortium called FANTOM consortium . The goaIn general, progress in understanding biology through RNA sequencing rests heavily on the shoulders of technological advancements. An enduring goal of the field is to increase the detection sensitivity in order to gain information when only small quantities of RNA or DNA are available. Within the last decades, substantial progress has been made on that front, making it possible to sequence the RNA content even from single cells. The field of single cell transcriptomics has subsequently greatly enhanced our understanding of cellular heterogeneity across many tissues . This leThis started to change with the first demonstration of single-cell sequencing of the small RNAs . With thBeyond simply probing which ncRNA molecules are present in any given sample, sequencing technology can also give insights into the functional role of ncRNAs. On the computational side, tools are being developed to help us to understand what the myriad of ncRNA sequences obtained by sequencing reveals about ncRNA function. Recent computational tools based on deep learning were developed to improve sequence classification into ncRNA classes or infer function of short ncRNAs transcripts based on the sequences alone ,104.One of the most studied roles for ncRNAs is the regulation of the transcriptome. This can take place at the different stages such as the transcriptional, post-transcriptional or chromatin level . Since tncRNAs have the ability to directly interact with the transcriptional machinery and thereby regulate gene expression. One of the most comprehensively studied examples of that is the mechanism of sex-chromosome dosage compensation . The inaIn contrast to transcriptional silencing, sequencing ncRNAs also led to important insights into transcriptional activation. One example for this is the discovery of eRNAs. Enhancers are genomic regions that activate transcription from a distance and are often marked by histone modifications such as H3K27Ac . InteresA major determinant of transcriptional regulation is the chromatin state . Hence, One of the first methods that aimed to map the global RNA-chromatin interactome in an unbiased fashion was named global RNA interactions with DNA by deep sequencing (GRID-seq) . GRID-seAnother set of methods that have been termed “hybridization capture methods” detect interaction sites of ncRNA with chromatin in a genome-wide fashion . MethodsBesides direct interaction with the proteins that bind to DNA, ncRNAs have also been shown to directly interact with DNA and thereby affect chromatin accessibility. One example of these direct interactions are triple helices called RNA–DNA–DNA triplexes . RecentlAnother form of RNA-DNA hybrid, which can involve mRNA as well as ncRNAs, resulting in the displacement of single-stranded DNA are so called R-loops. Mounting evidence accumulated in recent years suggests a number of regulatory functions of R-loops in gene expression . InitialOne of the most extensively studied ways to regulate the transcriptome post transcription is through miRNAs . AccordiBeyond the mere description of which miRNAs are expressed under any given circumstances, sequencing technology also showed how miRNAs regulate the transcriptome on a genome-wide scale. One technique that aims to accomplish this is called crosslinking, ligation, and sequencing of hybrids (CLASH) . CLASH bMore recently, advances in sequencing protocols enabled the investigation of the intracellular miRNA kinetics . The conBesides the above described roles of ncRNAs in the regulation of transcript production and translation, there is also a more indirect way in which ncRNAs can influence the transcriptome. lncRNAs, for example, have been shown to act as molecular scaffolds in cells . The lncAs transcriptional and posttranscriptional regulators of gene expression, ncRNAs are involved in many cellular physiological and pathological processes. Thus, they represent a promising target in medical research. In particular, RNA sequencing has uncovered many ncRNAs that are differentially expressed in the context of various diseases. Accordingly, ncRNA research has the potential to elucidate pathological mechanisms, identify novel prognostic and diagnostic markers or represent therapeutic targets . This poIn order to lay out the ncRNA landscape, sequencing approaches are needed to broadly uncover which ncRNAs are expressed in any given tissue. Thus, RNA sequencing greatly helped to unravel the transcriptomic landscape and thus generate reference atlases for comparison of pathological conditions . As an eIn general, there are large differences in the abundance of ncRNAs within different cell types and tissues. Especially circRNAs and lncRNAs exhibit tissue-specific expression ,138,139.Due to their complex regulatory functions, aberrantly expressed ncRNAs may contribute to the pathogenesis and progression of disease. This has been reported in the context of numerous pathologies in particular cancer  and neurOnce identified, sequencing of circRNAs can also enable insights into their potential function based on their sequence. According to the competing endogenous RNA hypothesis , circRNAInterestingly, in addition to these endogenous interactions between e.g., miRNAs and their mRNA targets, there are also interactions between pathogen-derived RNAs and human host RNAs. In a recent study, Yang et al. report that the genomic RNA of the SARS-CoV2 virus interacts with a number of host RNAs . Using sLiquid biopsy is a promising approach in clinical medicine that involves the collection of body fluids for diagnostic and prognostic analysis and monitoring. The aim is to replace more invasive tissue sampling and examinations as well as to enable earlier diagnosis and continuous monitoring especially for diseases that benefit from early detection. In order to find biomarkers for these purposes, RNA, DNA or proteins can be isolated and analyzed from cells found in e.g., blood, bronchial lavage, urine or cerebrospinal fluid. Especially for the investigation of neurological diseases, liquid biopsies offer a promising opportunity, given that the human brain is one of the most difficult organs to access for tissue biopsy in the human body. These benefits are exemplified in a recent study which analyzed small RNA sequencing data obtained from Parkinson’s disease patients’ whole blood . The autOne advantage of analyzing RNA from whole blood samples is that it is technically rather simple, yet very informative. A recent study has demonstrated that whole blood transcriptome predicts gene expression for other tissues in the body . InteresIn addition to these approaches, analysis of extracellular RNA emerges as a promising field for medical diagnostics. Sequencing cell-free RNA has demonstrated encouraging results for the diagnosis of several diseases ranging from cancer to cardiovascular diseases . In contAlongside advancements in the field of ncRNA research in human health and disease, in recent years there was also a surge in research of non-human ncRNAs and their potential roles. Thanks to new sequencing methods and novel approaches to data analysis, it has become possible to elucidate ncRNAs and their roles in viruses and various organisms ranging from archaea over scorpions to mice.Here, we provide a general overview of some of the recent advancements in research fields beyond human biology with the help of sequencing technology and computational analysis.Viral ncRNA studies are a rapidly evolving research area that was propelled by the rise of sequencing technologies . Since tMore recently, RNA-seq was used to assess the ncRNA repertoire produced by Ebola virus (EBOV) and Marburg virus. It was found that EBOV encodes small ncRNAs that act independently of the host miRNA machinery and do not display the ability to suppress viral replication or silence gene expression, thus leaving the question of their role in the host cell open .Besides human viruses, other viral pathogens have also been investigated. A recent study examined the African Swine Fever virus (ASFV), which lethally infects pigs going along with substantial economic ramifications ,183. SevSequencing has also opened up new horizons in prokaryotic ncRNA research. The development of metatranscriptomics has proven particularly useful, wherein the transcriptome of a complex microbiome is sequenced as a whole. This not only makes it possible to detect even lowly expressed genes, such as some ncRNAs but enables the investigation of non-culturable organisms using high-throughput sequencing ,185. TheAn important class of ncRNAs found in prokaryotes are small RNAs (sRNA) that range in length from 50–500 nt and regulate gene expression through RNA-RNA interactions . They weHalobacteria. Shotgun metagenomic sequencing was employed and resulted in the identification of over 1500 ncRNAs, 54% of which were of archaeal origin. Interestingly, significantly more intergenic sRNAs were found in archaea than in bacteria and the reverse was observed for antisense sRNAs [A recent study looked at the metatranscriptomic landscape of extremophiles inhabiting halite nodules  in the Atacama desert. Most of the extremophiles belonged to the domain of Archaea and specifically the class of se sRNAs .The rise of sequencing technologies has also enabled more specific and focused plant-based studies of ncRNAs. A particular focus has been laid on the role of ncRNAs in plant immunity, development and stress response  ,193. SeqGenerally, studies of plants used as crops have great economic significance and are therefore of particular value to society. Notably, crop species such as Chinese cabbage and rice have come under scrutiny recently. By employing NGS based approaches  the roles of ncRNAs in conferring resistance against infections ,199,200,Arabidopsis thaliana [A good example of a specialized sequencing approach being used in plant ncRNA research is the study by Ariel et al. where by employing CHiRp sequencing the function of lncRNA Auxin-Regulated Promoter Loop (APOLO) was studied in thaliana . The resthaliana .Further advancements in ncRNA research in plants have been made by the use of long-read sequencing technology. One example is the study of lncRNA expression in seed development of wheat and triticale (hybrid of wheat and rye) by nanopore sequencing. The study highlighted the utility of nanopore sequencing for genomes with high complexity and identified a large number of novel lncRNAs . In the Finally, considering the characteristics specific to plant lncRNAs ,205,206 Saccharomyces cerevisiae), one of the most studied fungi species, with many ncRNAs acting as important gene expression regulators [ncRNAs account for up to 25% of the genome of budding yeast  took advantage of readily available sequencing data. By combining datasets with data generated by themselves, the authors were able to identify more than 10,000 novel lncRNAs in A. albopictus including ncRNAs with potentially modulating effects on Dengue and Zika virus replication [Invertebrates comprise ~95% of animal species , and alm elegans . RNA seqfly wing . This ncfly wing . Similarlication . StudiesAndroctonus crassicauda [Another recent study investigated the poorly defined lncRNA landscape of scorpions, in particular, the venom glands of the species ssicauda . In the ssicauda . This stIn comparison to other taxa, the state of ncRNA research is more advanced in vertebrates. Mammalian organisms, in particular rodents and humans, have been studied extensively. But other vertebrates have traditionally not been the focus of vertebrate research. Examples include the study of the repertoire and function of circRNAs in the liver of the Whitespotted Bamboo shark , and theAmongst avians, chicken has been extensively researched. Studies utilizing sequencing technologies have focused on the roles ncRNAs play in embryogenesis , environStudies focused on livestock represent another example of understudied vertebrate research given its large economic impact. An example is the investigation of ncRNAs in cattle considering that cattle and buffalo meat and milk are the sources of 45% of global animal protein supply . ComputaBeyond tackling food supply issues for a growing world population, the generation of transgenic animals for production of biopharmaceuticals presents another incentive for increased research efforts in vertebrates. One of the main methods used to this end is somatic nuclear cell transfer (SCNT) , which sHomo sapiens. By analyzing DNA sequencing data from Homo sapiens, Neanderthals and chimpanzees, the authors found that chimpanzees had no lncRNAs amongst the genes that have an association with modern human personality and a much higher proportion of lncRNAs was detected in Homo sapiens compared to Neanderthals. In total, 94% of those genes found only in Homo sapiens were ncRNAs, most being lncRNAs [Interestingly, the increasingly broad application of sequencing to discover the ncRNA landscape of traditionally understudied species is not limited to species that are alive today. Ancient DNA sequencing offers insights into genomic landscapes of extinct life forms . A recen lncRNAs .Although most of the above described findings were based on short-read sequencing technology, the development of long-read sequencing has also left its mark on mammalian research. For instance by employing nanopore sequencing, human and mouse brain circRNAs were characterized, revealing widespread splicing events in circRNAs, highlighting the importance of developing sequencing technologies . It willThe rise of sequencing technologies has revolutionized the study of the transcriptome, including ncRNAs. Much has been understood about ncRNAs and recent years have seen a rapid increase of published ncRNA papers . Thanks The development of single-cell sequencing has allowed the study of ncRNAs at the single-cell level and with the more specialized and targeted sequencing methods it has become possible to study not only the landscape of ncRNAs but also their functionalities in the cell. The recently developed long-read sequencing technology demonstrated its potential for the field of ncRNA research, for example by elucidating circRNA exon composition . These tAlthough the field of ncRNA sequencing has come a long way since the discovery of the first ncRNAs in the 1950s and 1960s, exciting technological advances will continually advance our understanding of the complexity of the ncRNA transcriptome. For instance, the performance of nanopore sequencing technology has been steadily improving in the last years . The abiin vivo. Detection of ncRNAs still remains challenging. Reasons for this include the low expression levels of many ncRNAs [Nevertheless, challenges remain. With the discovery of novel ncRNA species, increased efforts will be needed to elucidate their functions y ncRNAs , unique y ncRNAs . Due to y ncRNAs , Thomas y ncRNAs  and Warry ncRNAs . Similary ncRNAs ,252. Anny ncRNAs . The laty ncRNAs ,255. Fur


Pichia pastoris expressing LMBV major capsid protein (MCPD). The recombinant GS115-pW317-MCPD was then used to immunize largemouth bass via oral administration, and mucosal immune response mediated by immunoglobulins (Igs) was measured after oral immunization. Serum antibody levels were measured by ELISA, neutralizing antibody titers were determined by serum neutralization test (SNT), antigen presentation-related gene expressions were detected by RT-PCR, and the histopathological characteristics of immunized fish were assessed after challenging with 0.1 ml 107.19 TCID50/ml LMBV. The relative percentage survival (RPS) was also determined. Our results showed that the serum antibody titers of immunized fish were significantly higher than that of control groups (P < 0.05). IgT and IgM expressions in gut were increased significantly after vaccination with GS115-pW317-MCPD; however, much stronger response in gut was observed as compared with gill. The expression levels of major histocompatibility complex (MHC) II, CD8, and T-cell receptor (TCR) were significantly elevated in GS115-pW317-MCPD group (P < 0.05), while CD4 and MHC I transcription levels remained unchanged after oral immunization (P > 0.05). The RPS of fish orally immunized with 1.0 × 108 CFU/g GS115-pW317-MCPD was reached up to 41.6% after challenge with 0.1 ml 109.46 TCID50/ml LMBV. Moreover, orally immunizing with GS115-pW317-MCPD can relieve the pathological damage caused by LMBV. Therefore, GS115-pW317-MCPD showed a promising potential against LMBV.Largemouth bass iridovirus (LMBV) can cause high mortality and lead to heavy economic loss in the cultivation of largemouth bass, but there was no effective treatment. Here, the present study constructed a recombinant  Micropterus salmoides) was a widely cultured freshwater fish in China   , 13, whiPichia pastoris GS115 was used as a host to express the LMBV major capsid protein (MCP) and introduced into P. pastoris GS115 by electroporation. Then, orally immunized to largemouth bass. Serum antibody levels of fish immunized with recombinant GS115-pW317-MCPD were measured by ELISA, neutralizing antibody titers were determined by SNT, and immune response of immunoglobulins (Igs) and antigen presentation-related gene expressions were detected by RT-PCR. The relative percentage survival (RPS) and the histopathological characteristics of immunized fish were also assessed.Yeast is an ideal protein preparation platform; it was easy to culture and suitable for large-scale production. Additionally, β-glucan, the main compound of the cell wall of yeast, was recognized as an immune-stimulant supplement in fish, which made the yeast an attractive candidate for antigen delivery to the intestinal mucosa . On the -1; nitrites < 0.02 mg l-1; ammonia < 0.1 mg l-1.Healthy largemouth bass (24.6 ± 3.1 g) were purchased from Zhejiang Institute of Freshwater Fisheries Comprehensive Experimental Base  and were acclimatized in recirculating aquaculture system for 14 days. Before the experiment, fish were randomly sampled for the examination of bacteria, parasite (microexamination), and virus (PCR for LMBV); no pathogens were detected, and no naturally dead fish were found during the temporary cultivation. Water quality conditions were controlled by recirculating the aquaculture system as follows: temperature: 28°C ± 1°C, dissolved oxygen >5 mg lP. pastoris GS115 was obtained from Hangzhou Fenghai Biotechnology Co., Ltd.The LMBV was originally isolated and identified from diseased largemouth bass by our laboratory. Fathead minnow (FHM) cell was obtained from Pearl River Fisheries Research Institute and grown at 28°C in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. Epithelioma papulosum cyprini (EPC) cells were maintained in our lab and grown at 28°C in DMEM. The MCPD protein were analyzed and optimized (see in the attachment). In order to facilitate the purification of protein and antibody level determination, hexa-histidine tag was designed. The MCPD gene (hexa-histidine tag at the N-terminus) was synthesized by Sangon Biotech  Co., Ltd.Major epitope regions of MCPD were listed as follows: F : AATTGGTTTGACTAATTCCATAAT; R : AATGTTCGTCAAAATGGTGAC. EcoR I and NotI sites were also added, respectively  medium at 37°C.The primers for amplifying ectively Figure 1MCPD cells were harvested and then disrupted by sonication. The protein concentration of the culture lysates was then determined. The MCPD proteins were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and then recombinant MCPD proteins (with His-Tag) were identified by Western blotting using anti-His-tag monoclonal antibody and Horseradish Peroxidase (RRP)-conjugated goat anti-mouse IgG as antibodies .After induction for 24 h, recombinant GS115-pW317-8 CFU/g GS115-pW317-MCPD , 107 CFU/g GS115-pW317-MCPD , GS115-pW317 (108 CFU/g and 107 CFU/g), and PBS . Briefly, the harvested GS115-pW317-MCPD and GS115-pW317 were washed twice with PBS, and then the concentrations of harvested GS115-pW317-MCPD and GS115-pW317 were determined, and the required amount of GS115-pW317-MCPD and GS115-pW317 was mixed with commercial basal diet for oral immunization. All fish were fed twice for 1 day at 08:00 and 16:00 h, respectively. The feeding trial was conducted for 2 weeks. Sera were collected from the caudal vein of each fish for antibody titer determination. Gill, gut, and kidney were sampled on days 7, 14, 21, 28, and 35 for immune response and gene expression assays (3 fish/time point). All treatments and control groups were conducted with three replicates (30 fish for each).Five groups of fish (30 each) were orally immunized with 102CO3/NaHCO3 buffer (pH 9.6) at 37°C for 4 h . Purified MCPD protein (with His-Tag) was then added into 96-microtiter plate at 37°C; after incubation for 1 h, each well was washed with Phosphate Buffered Saline with 0.05% Tween (PBST) 3 times. Here, 100 µl of McAb Mouse Anti-His Tag  used as primary antibody were then added into each well of plate at 37°C for 1 h, then washed with PBST buffer 3 times. Subsequently, HRP-conjugated goat-mouse IgG antibody  used as secondary antibody was added into each well of plate at 37°C; after incubation for 1 h, each well was washed by PBST buffer 3 times, then 2 mol/L H2SO4 were used to stop the reaction; Tetramethylbenzidine (TMB) was used to color the result.The antibody levels of immunized fish were determined by ELISA according to previous studies , 10. ThrGene expression levels of IgM and IgT after immunization were conducted. Total RNA extracted from each tissue  was prepared by TRIzol lysis . cDNA was then synthesized according to the protocol of Reverse Transcriptase M-MLV Kit . The RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix Kit . The primer was designed following previous reports , 16.MHC) I, MHC II, CD8, CD4, and T-cell receptor (TCR) were also determined by RT-PCR. Briefly, total RNA was extracted from gill, gut, and kidney by TRIzol Reagent . The RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix Kit  and carried out in a Stratagene MxPro System  in 96-well reaction plates.Expression levels of antigen presentation-related genes major histocompatibility complex  were added to EPC cell monolayers. Finally, 5 days after incubation, the highest dilution at which 50% EPC cells were inhibited was considered as the LMBV neutralizing antibody titer.Neutralizing antibody titers were determined by serum neutralization test (SNT) in EPC cells according to previous methods . FirstlyMCPD and GS115-pW317 and injected 0.1 ml 108.66 TCID50/30 ml LMBV per fish, six largemouth bass were randomly selected from PBS group, three fish were challenged with 0.1 ml 107.19 TCID50/ml LMBV, the remaining three fish were treated with no virus. After 14 days, the liver, kidney, and spleen samples in each group were received, fixed in 10% formalin, embedded in paraffin wax, and sectioned by microtome, and then hematoxylin and eosin (H&E) staining was conducted to analyze the histological change of immunized fish.At 35 days post immunization (dpi), three largemouth bass were randomly selected from group GS115-pW317-MCPD  and GS115-pW317-MCPD . GS115-pW317 (107 CFU/g feed and 108 CFU/g) and PBS  served as control group. All fish were fed twice a day and lasted for 2 weeks. On 21 dpi, all fish were challenged with 0.1 ml 109.46 TCID50/ml LMBV per fish. Mortalities in each group were recorded daily for 15 days. The RPS was calculated as follows:Five groups were set in challenge test. Each group contains 20 largemouth bass; each group was performed with three replicates. Immunized groups were orally immunized with GS115-pW317-The data were expressed as the mean and standard deviation (mean ± SD) and analyzed by Statistical Product and Service Solutions (SPSS 19.0). The difference was considered statistically significant when P < 0.05 and highly significant when P < 0.01.MCPD (P. pastoris GS115 by electroporation. The cell lysates GS115-pW317-MCPD and GS115-pW317 were subjected to SDS-PAGE and Western blot. A specific band of about 43 kDa was detected in GS115-pW317-MCPD (MCPD was successfully expressed in P. pastoris GS115.GS115-pW317-MCPD-immunized group were significantly higher than those of fish fed GS115-pW317 or PBS (P < 0.01). Serum antibody levels in 108 CFU/g GS115-pW317-MCPD-immunized group were about 8-fold higher than those in the PBS group and GS115-pW317 group .Compared with GS115-pW317 and PBS control groups, IgT was highly expressed in head kidney and gut. In head kidney, the IgT gene expression reached its peak at 21 dpi, which was about 12-fold higher than that in the control PBS group; the corresponding data in gut was 5.5-fold. However, much stronger response in gut was observed as compared with gill after oral immunization Figure 3MHC-I mRNA in the tested tissues were increased as compared with those in the control group after oral immunization. In gill, the expressions of MHC-I mRNA in GS115-pW317-MCPD-immunized group were significantly upregulated and reached the top at 21 dpi, which was 2.4-fold higher than those in the PBS groups . In kidney, the expression level of MHC II reached up to a peak at 21 dpi, which was 2.4-fold higher than that in the PBS group. In gut, the peak value of 108 CFU/g group was found at 21 dpi, which was 4.5-fold higher than that in the PBS group .The results of mRNA expressions of CD8 in kidney, gut, and gill were significantly increased as compared with those in the control group (P < 0.01) (CD8 mRNA were significantly upregulated at 28 dpi as compared with those in the PBS group (P < 0.01)  Figure 4TCR mRNA in the three tested tissues were increased as compared with those in the control group. The peak value of kidney tissue was 2.6 times higher than that of the PBS group on 28 dpi (P < 0.01). In gill, the expressions of TCR mRNA reached up to a peak at 35 dpi, with a peak value of 2.2-fold higher than that of the PBS group . However, the pathological injuries in spleen and kidney were relatively light when immunized with 108 CFU/g GS115-pW317-MCPD.Varying degrees of cytopathic lesions were detected in largemouth bass after challenging with LMBV, however, in the control group treated with non virus, cells were normal with intact structure, and no pathological changes were found   Figure 9MCP protein of LMBV was constructed via the yeast P. pastoris, and the results from the challenge test showed that GS115-pW317-MCPD can effectively elicit immunity response and protect largemouth bass against LMBV, which indicated that P. pastoris could become a promising oral vaccine carrier against fish virus.LMBV was one of the most harmful viruses in the cultivation of largemouth bass. However, there is still no effective drug to treat LMBV infection. Vaccination is considered as an effective method to control the outbreak of fish disease . The rouMCPD was 41.6%. However, Yi et al.  were one of the most studied invasion-related proteins; it could invade mammalian cells by binding to the α5β1 integrin on the surface of the host cell membrane. Therefore, it was usually used to carry DNA to host cells  as a live bacterial vector to coexpress Trichinella spiralis SS1 and murine interleukin-4; in vivo results exhibited that FnBPA increased the effect of antigen presentation and immune protection. Additional studies are required to further coexpress more antigens; some adjuvant or invasion-related proteins are needed to enhance the humoral and cellular immunity   in turbot and flounder was detected in gill and spleen  , 29. Mor mykiss) , 29. IgM mykiss) . The expn of IgM . As for d spleen . Becaused spleen . Our resd spleen , 29. BasMHC I and II, antigens are processed and presented to specific lymphocytes .MCPD. LH, HD, LL and ZY contributed significantly to analysis and article preparation. WY performed the data analyses. XP helped evaluate the immune efficacy. All authors contributed to the article and approved the submitted version.JY, GY, WZ and HZ contributed to the conception of the study. JY, XY, CZ constructed the recombinant GS115-pW317-The research was supported by the National Key R&D Program of China (2019YFD0900104), the key project program of Huzhou City , and “San Nong Liu Fang” Science and Technology Collaboration Projects (2020SNLF020).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Zika virus (ZIKV), without a vaccine or an effective treatment approved to date, has globally spread in the last century. The infection caused by ZIKV in humans has changed progressively from mild to subclinical in recent years, causing epidemics with greater infectivity, tropism towards new tissues and other related symptoms as a product of various emergent ZIKV–host cell interactions. However, it is still unknown why or how the RNA genome structure impacts those interactions in differential evolutionary origin strains. Moreover, the genomic comparison of ZIKV strains from the sequence-based phylogenetic analysis is well known, but differences from RNA structure comparisons have barely been studied. Thus, in order to understand the RNA genome variability of lineages of various geographic distributions better, 410 complete genomes in a phylogenomic scanning were used to study the conservation of structured RNAs. Our results show the contemporary landscape of conserved structured regions with unique conserved structured regions in clades or in lineages within circulating ZIKV strains. We propose these structures as candidates for further experimental validation to establish their potential role in vital functions of the viral cycle of ZIKV and their possible associations with the singularities of different outbreaks that lead to ZIKV populations to acquire nucleotide substitutions, which is evidence of the local structure genome differentiation. Aedes genus [Zika virus (ZIKV) was first identified in Rhesus monkeys in the Zika forests of Uganda in 1947. Its spread from Africa reached Southeast Asia, limiting its associated symptoms to feverish symptoms, conjunctivitis and joint pain during the 20th century ,4. Declaes genus .Flavivirus genus [ZIKV is a single-stranded positive-sense RNA arbovirus within the us genus , the genus genus . The genus genus , and a cus genus .The ZIKV genome folds in a secondary RNA structure shape like RNA-type molecules which also occurs in other RNA viruses, where these structures perform pivotal functions during the viral cycle, such as regulating translation, promoting replication and evading host cell antiviral responses ,11,12. FHowever, patterns of RNA secondary structures in viruses with RNA genomes, despite their importance, have been poorly studied  in a comA graphical scheme of rational design of materials, methods and results can be seen in An in-depth search for complete Zika virus genomes was performed in NCBI and VipR databases (search date: July 2020) to retrieve a total of 1023 genomes ,25. A loAll multiple genomic alignments were performed by Clustal Omega v1.2.4 , settingThe phylogenetic analysis was based on the whole genomic sequences (including both CDS and UTR regions), and the following R packages were used to evaluate genomic sequence relationships: APE, seqinr and Phangorn ,32,33. AFinally, to describe the variability and conservation of sequences, percentages of pairwise identity and the number of identical sites per nucleotide columns of the alignment were calculated using Geneious Prime v2020.1 . From th−27.In order to analyze the geographic similarity of the RNA conserved structures, RNAz v2.1.1 software  was emplFinally, as a statistical support of the secondary RNA structures, two different randomization alignment routes were performed: general  and specific (to each window). In the general randomization, 100 randomized alignments from each geographic or subgeographic region were obtained . These were analyzed entirely in RNAz, maintaining the size of the sliding window, Z value and P-value parameters. The relative false positive detection rate (FP) and the percentage of specificity (% Specificity) were determined. The specific randomization  per each of the windows detected as structured in the original alignment was generated, and they were analyzed with the same RNAz parameters mentioned above, removing from the analysis those windows that had a rate of false positives greater than 0.05 (% FP > 0.05).The NJ approach shows the diverse relationships between the viral genomes and the continents A. A grouThe circular design of the cladogram was changed to a traditional design B to provRegarding Maximum Likelihood (ML) phylogenetic trees , these aIn the descriptive analysis of sequence variability, summarized in From the ANOVA analysis, significant differences in the choice of window size were found , and a specificity index higher than 95% in all cases; therefore, the filters used to run the pipeline of RNAz  were effective in selecting information of the detected structures, and the results were statistically significant . In the The results of the most representative secondary RNA structures at the inter-geographical region level are found in On the other hand, RNA structures that overlapped with RNA models from RFAM are shown in The findings here reported in both phylogenetic trees agree with the historical records of Zika virus outbreaks. The sequences from Africa are basal, as a viral origin, followed by Asia and last America . AccordiAdditionally, the grouped sequences in the clades of both types of cladograms reflect the establishment of individual viral genotypes in geographically delimited regions, regardless of the methodology used. We always found the Colombia clade, the Caribbean clade, the Mexico clade, Asia Southeast, Asia Continental, and Africa to have good support in the NJ analysis. These groups agree with other phylogenetic trees reported in the literature with a set of sequences previously reported ,40.Culex spp, has a greater vector-specific viral diversity in contrast to the variety found in the host vertebrate [The global and variants from specific geographic regions full-length sequences were also analyzed using comparative genomics to detect conserved secondary structures to show the contemporary landscape of conserved structured regions with unique conserved patterns. The high degree of homology seen between the Zika viral sequences from distant geographic regions can be contextualized with similar features found in other flaviviruses, for instance, the similarity within Dengue serotypes, where its most variable fragment, the 3 ’UTR region, reaches 97% of pairwise identity . Therefortebrate .Flavivirus genus, especially in structures that were also found in the RFAM database [Aedes africanus. It is acknowledged that secondary RNA structures are vital factors in flaviviruses for viral replication in their respective disease-transmitting insects [Regarding the conserved structured regions of RNA in all the Zika virus sequences, at the 5’ and 3’ UTR ends, several of their essential functions have been reported in the database ,45. Thusdatabase . Additiodatabase ,47. Likedatabase . The dif insects . For exa insects .Culex spp., but it survives in its salivary glands; therefore, it is a key structure to complete the viral cycle, from the ingestion of blood to transmission by mosquito saliva [Culex spp. insects [Another example is a WNV mutant which lacks the formation of the sfRNA1 structure, and it does not survive in the intestine of the insect o saliva . This is insects . The virWith respect to the structural region of RNA shared among Asia, Oceania, and America in the position close to 1.1 kb, it is found to be intriguing because it has been experimentally validated, in vivo, and its importance in the function of the Zika virus has been reported . The autFinally, the RNA structures found in common between America and Oceania might contribute to the genomic particularity present in the outbreaks, where the tissue damage and viral infectivity were superior in contrast to Asian and African lineages in experimental studies with mice . AdditioPerforming this comparative analysis between Zika virus genomes and their RNA conserved secondary structures allowed the selection of regions and certain specific structures, which are distinguished by their patterns in genomic comparison at the inter-geographical lineage level. In this way, these patterns of structural conservation guided the selection of potential functionally relevant structures in the viral cycle of ZIKV. Further experimental analysis for associating new functions must be performed. In the future, these structures may have the potential to be targeted to negatively affect viral replication .