Untranslated Regions of a Segmented Kindia Tick Virus Genome Are Highly Conserved and Contain Multiple Regulatory Elements for Viral Replication

Novel segmented tick-borne RNA viruses belonging to the group of Jingmenviruses (JMVs) are widespread across Africa, Asia, Europe, and America. In this work, we obtained whole-genome sequences of two Kindia tick virus (KITV) isolates and performed modeling and the functional annotation of the secondary structure of 5′ and 3′ UTRs from JMV and KITV viruses. UTRs of various KITV segments are characterized by the following points: (1) the polyadenylated 3′ UTR; (2) 5′ DAR and 3′ DAR motifs; (3) a highly conserved 5′-CACAG-3′ pentanucleotide; (4) a binding site of the La protein; (5) multiple UAG sites providing interactions with the MSI1 protein; (6) three homologous sequences in the 5′ UTR and 3′ UTR of segment 2; (7) the segment 2 3′ UTR of a KITV/2017/1 isolate, which comprises two consecutive 40 nucleotide repeats forming a Y-3 structure; (8) a 35-nucleotide deletion in the second repeat of the segment 2 3′ UTR of KITV/2018/1 and KITV/2018/2 isolates, leading to a modification of the Y-3 structure; (9) two pseudoknots in the segment 2 3′ UTR; (10) the 5′ UTR and 3′ UTR being represented by patterns of conserved motifs; (11) the 5′-CAAGUG-3′ sequence occurring in early UTR hairpins. Thus, we identified regulatory elements in the UTRs of KITV, which are characteristic of orthoflaviviruses. This suggests that they hold functional significance for the replication of JMVs and the evolutionary similarity between orthoflaviviruses and segmented flavi-like viruses.


Introduction
Over the past five years, many novel Flaviviridae viruses possessing atypical genome structures have been discovered using high-throughput sequencing, which has challenged the traditional principles of virus classification [1].The Flaviviridae family comprises epidemiologically significant orthoflaviviruses, such as West Nile virus, Zika virus, dengue virus, yellow fever virus, tick-borne encephalitis, etc. Jingmenviruses (JMVs) are novel RNA viruses that were first discovered in Rhipicephalus microplus ticks from the Jingmen region of Hubei Province in China in 2014 [2].According to the International Committee on Taxonomy of Viruses (ICTV), the JMV group is considered to comprise unclassified viruses of the Flaviviridae family and currently includes Jingmen tick virus (JMTV), Mogiana tick virus (MGTV), Kindia tick virus (KITV), Alongshan virus (ALSV), Yanggou tick virus (YGTV), Takachi virus (TAKV), Harz mountain virus (HMV), Sichuan tick virus (SCTV), SCWL tick virus (SCWLTV), etc.The genetic material of these viruses has been found not only in ticks but also in mosquitoes, cattle, bats, rodents, and humans [3][4][5][6].To date, JMVs have been discovered in Asia, Europe, Central and South America, and Africa.Recently, the discovery of JMVs in Russia and their potential pathogenicity to humans have been first reported [7][8][9].JMVs are a new group of viruses that bear a risk of causing a pandemic.Because they are closely related to arthropods, they are found in animals in close contact with humans and cause febrile illness in patients.
JMVs are fundamentally different from "classical" orthoflaviviruses due to a segmented single-stranded RNA positive-sense genome.The genome consists of four (or five) segments.Each segment comprises one or more open reading frames (ORFs) and characteristic 5 ′ and 3 ′ untranslated regions (5 ′ and 3 ′ UTRs) and is presumably packaged into a separate viral particle [4,10].The proteins encoded by segments 1 and 3 are genetically and functionally related to nonstructural NS3 and NS5 proteins of classical orthoflaviviruses from the Flaviviridae family.The RNA-dependent RNA polymerase gene was shown to be integrated into the genome of Ixodes ricinus ticks.The other two segments, encoding structural VP1-VP3 proteins, are of unknown origin [11].Very little information is currently available on the secondary structure of the JMV 5 ′ and 3 ′ UTRs that function as cis-acting elements during viral genome replication, translation, and viral life cycle regulation [6,12].Knowledge of the structure of JMV 5 ′ and 3 ′ UTRs will help us to study the endemic potential of JMVs and to develop antiviral drugs and vaccines.
Kindia tick virus (KITV) is a novel unclassified tick-borne flavi-like virus from the JMV group of the Flaviviridae family.It was first discovered in ixodid ticks Rhipicephalus geigyi collected from cattle around the city of Kindia, Republic of Guinea, in 2017 [13].Then, KITV genetic material was found in six more Rhipicephalus geigyi ticks from the 2021 collection [14].KITV has a genome structure typical of that in JMVs; NS3 and NS5 proteins are structurally and functionally similar to those in orthoflaviviruses, which confirm their possible evolutionary relationship and taxonomic unity; and structural VP1-VP3 proteins of KITV have no analogues among known viral proteins [15].In this study, the secondary structure of the KITV RNA 5 ′ and 3 ′ UTRs was for the first time modeled and analyzed, regulatory elements in the KITV 5 ′ and 3 ′ UTRs characteristic of orthoflaviviruses were discovered, and conserved motifs were identified in JMVs, including KITV.

Tick Collection
The collection of ixodid ticks was formed in 2018 from freshly slaughtered cattle in a slaughterhouse in Kindia, the Republic of Guinea.Sampled ticks were classified into species based on their morphological characteristics.Next, they were homogenized and stored at −80 • C until analyses were undertaken [16].Tick collection was kindly provided by the Research Institute of Applied Biology of the Republic of Guinea.

Whole-Genome Sequencing
Total RNA was isolated from tick (Rhipicephalus spp.) homogenates using phenolchloroform extraction and an ExtractRNA reagent (Evrogen, Moscow, Russia).Reverse transcription was performed using a MMLV RT kit (Evrogen, Moscow, Russia).Screening for the genetic material of segmented flavi-like viruses was carried out through a PCR using JMV_f-TGGACCAGGGCMGTIGGRGAGTA and JMV_r-GAAAACCTGRTAGTYIGGGT CGCA oligonucleotides [2].Kindia tick virus cDNA fragments were amplified using a Q5 High-Fidelity DNA Polymerase kit (NEB, Ipswich, MA, UK), and original oligonucleotides were calculated by the authors.The data are shown in Table S1.Amplicons were analyzed through electrophoresis in 2% agarose gel and purified using a Cleanup Standard kit (Evrogen, Moscow, Russia).The sequencing reaction was performed using a BigDye Terminator v3.1 kit (Thermo FS, Waltham, MA, USA).After the sequencing reaction, fragments were purified through direct reprecipitation with ethanol.Wholegenome sequencing was performed using an ABI 3500/3500xl device (Applied Biosystems, Waltham, MA, USA).Whole-genome sequence assembly and sequence chromatogram processing were performed using Lasergene 10 SeqMan (DNASTAR, Madison, WI, USA) and UGENE version: 1.31.1 (UNIPRO, Moscow, Russia) software.The search for open reading frames and translation into amino acid sequences were performed using Vector NTI (Invitrogen, Waltham, MA, USA).All whole-genome sequences were deposited in the GenBank database under accession numbers MW341206-MW341213.
For the construction of a phylogenetic tree, complete nucleotide sequences of the segment 1 open reading frame from seventeen representative isolates of segmented flavilike viruses were aligned and analyzed through the maximum likelihood method with 1000 bootstrap replicates using the MEGA X molecular evolutionary genetic analysis software (PSU, Philadelphia, PA, USA).The tree optimization algorithm and distance correction (G + R + I) were selected using the JMODELTEST software version: 2.1.10(University of Vigo, Vigo, Spain).Isolates in the phylogenetic tree were labeled using a GenBank number followed by the isolate's name, country, origin, and year of isolation.

5 ′ UTR-3 ′ UTR Sequences and Structures
The primary criterion for the selection of untranslated regions was their size.Isolates lacking an untranslated region or that were shorter than 35 nucleotides, which indicated their under-sequencing, were excluded from the sample.The threshold was empirically chosen.
Secondary structures of the KITV RNA 5 ′ and 3 ′ UTRs of segments 1 and 2 were predicted using three independent tools as follows: ViennaRNA Fold (http://rna.tbi.univie.ac.at, accessed on 28 December 2023), UNA MFOLD 3.6 (http://www.unafold.org/mfold, accessed on 28 December 2023), and RNAstructure (https://rna.urmc.rochester.edu,accessed on 28 December 2023) [17][18][19].Simulation was performed at a folding temperature of 37 • C and under ionic conditions of 1 M NaCl without divalent ions.The parameters of "maximal distance between paired bases" (MDBPB) and "percent suboptimality" (%S) were selected manually.An MDBPB of 60 to 100 and a %S of up to 50% were established.The upper bound on the number of computed folds and the upper bound on the total number of single-stranded bases that are allowed in a bulge or interior loop were set at 25.Other parameters were set as default, and the initial free energy ∆G was set as the minimum value.For detailed analysis, secondary structures were linearized using the VARNA 3.9 software (http://varna.lri.fr,accessed on 28 December 2023) and redrawn using graphic editors [20].
in a bulge or interior loop were set at 25.Other parameters were set as default, and the initial free energy ∆G was set as the minimum value.For detailed analysis, secondary structures were linearized using the VARNA 3.9 software (http://varna.lri.fr,accessed on 28 December 2023) and redrawn using graphic editors [20].

KITV 5 ′ and 3 ′ UTR Sequences
Multiple alignment of KITV and MGTV 5 ′ and 3 ′ UTRs revealed that lengths of the 5 ′ and 3 ′ UTRs varied significantly due to various insertions, deletions, and nucleotide substitutions.The data are shown in Table 1.KITV is characterized by a short 5 ′ UTR (91-154 b.p.) and a longer 3 ′ UTR (up to 387 b.p.), which are consistent with the length of untranslated regions in Zika virus and tick-borne encephalitis virus.The data are shown in Table 1.The 3 ′ UTR of KITV and MGTV contains a ~20 b.p. polyA tail, which is not typical of viruses of the Flaviviridae family.Moreover, the 3 ′ UTR of all KITV segments was found to harbor multiple UAG sites (two to six) for interaction with the RNA-binding protein Musashi-1 (MSI1) associated with Zika virus neurotropism.The data are shown in Table S3.Note: *-the region is completely under-sequenced.

KITV Segment 1's 5 ′ and 3 ′ UTR Structures
The secondary structure of segment 1's 5 ′ UTR is represented by the two stem loops of SL-1 and SL-2.The model of the secondary structure is shown in Figure 2a.Hairpin 1 comprises a 5 ′ -GUGC-3 ′ inverted sequence that comprises the La autoantigen binding site [21].The CCAGG sequence is located 111 nucleotides downstream of the AUG start codon.In orthoflaviviruses, this sequence is called 5 ′ DAR.In the 3 ′ UTR, the complementary 5 ′ -CCUGG-3 ′ sequence (3 ′ DAR) was found 50 nucleotides downstream of the stop codon.The 5 ′ DAR-3 ′ DAR regions are involved in flavivirus genome cyclization [22].A distant location of the KITV 5 ′ DAR results in the emergence of three stem loops (SL-3, SL-4, SL-5) and a Y-structure.The 3 ′ UTR topology is conserved among the JMV isolates examined.This is represented by the Y-1 structure and SL-1 in all the KITV isolates and the MGTV/V4/11 isolate.The model of the secondary structure is shown in Figure 2b.A difference was found in the MGTV Yunnan2016 isolate in which this region was represented by two Y-structures.A highly conserved orthoflaviviral 5 ′ -CACAG-3 ′ pentanucleotide was found in hairpin 3 of the Y-1 structure.

KITV Segment 2's 5′ and 3′ UTR Structures
The secondary structure of segment 2's 5′ UTR is represented by SL-1 and SL-2.The model of secondary structure is shown in Figure 3a.This segment comprises the La binding region at position 12 and downstream regions 5′ DAR-3′ DAR.The 5′ DAR is located 50 nucleotides downstream of AUG, which results in SL-4 and an extended free end.The AUG start codon occurs in SL-4, and the 5′ DAR is located in the free end.Similarly, the 3′ UTR comprises the 3′ DAR at position 2608 of the SL-6 nucleotide structure.

KITV Segment 3's 5′ UTR Structure
The secondary structure of segment 3's 5′ UTR is represented by one stem loop and one Y-structure.The model of the secondary structure is shown in Figure 4. Hairpin 1 contains an inverted sequence of the La autoantigen binding site.The 5′-CCAGG-3′ sequence occurs 165 nucleotides downstream of the AUG start codon.No complementary 5′-CCTGG-3′ sequence (3′ DAR) was found in the 3′ UTR.The distant location of the 5′ DAR in KITV modifies the Y-structure through the emergence of four hairpins in it.The 5′-CACAG-3′ pentanucleotide was not found in the 3′ UTR, which may be due to the under-sequencing of this region.However, a similar sequence was found in the 5′ UTR of the KITV/2018/1 and KITV/2018/2 isolates (coordinates 12-16); in the KITV/2017/1 isolate, there was a substitution at position 14, leading to the disappearance of this pentanucleotide.The data are shown in Table S4.

KITV Segment 4's 5′ and 3′ UTR Structures
The secondary structure of segment 4's 5′ UTR is represented by SL-1, SL-2, and SL-3, and that of segment 4's 3′ UTR is represented by two Y-structures.Models of secondary structure are shown in Figure 5.The 5′ UTR SL-1 structure comprises the La binding region and the 5′-UGGCAAGUGC-3′ R1 sequence previously found in KITV segment 2's 5′ and 3′ UTRs.The same R1 sequence is present in the second hairpin of the 3′ UTR Y-1 structure.No other regulatory elements were found.

Discussion
Orthoflaviviruses are important pathogens that cause serious infections in humans, from mild fever to encephalitis and hemorrhagic fever, which often result in death.JMVs are a recently discovered group of viruses of unknown pathogenicity.They have a segmented RNA genome consisting of four segments, two of which are functionally related to the non-structural genes of viruses from the Orthoflavivirus genus, suggesting the existence of segmented flavi-like viruses in the Flaviviridae family.
In this study, we performed whole-genome sequencing of two new KITV/2018/1 and KITV/2018/2 isolates from the collection of Rhipicephalus spp.ticks from the city of Kindia, Republic of Guinea.The genomic analysis revealed that the KITV isolates clustered together with the JMTV_1 (MH155892) isolate and the MGTV/V4/11 (JX390986) isolate, which were previously detected in Rhipicephalus microplus ticks from the Central-West and Southeast Regions of Brazil and had 90% and 92% nucleotide sequence identity in ORF segment 1 [23].Interestingly, the KITV isolates from the Republic of Guinea and the JMTV and MGTV isolates from Brazil cluster within a monophyletic clade that is different from that of the isolates identified in Asia, Africa, and Southeast Europe, suggesting that these isolates likely belong to a novel virus species within JMVs.
Comparative analysis of the KITV genome and those of other flaviviruses revealed important structural elements of the 5 ′ and 3 ′ UTRs.The 5 ′ and 3 ′ UTR of segments 1-3 harbor functional orthoflaviviral 5 ′ DAR and 3 ′ DAR regions that are responsible for longrange RNA-RNA interactions during genome cyclization before replication onset [22,24].
The distant location of the 5 ′ DAR in segment 1 of the KITV genome leads to the emergence of SL-3, SL-4, SL-5, and a Y-structure; additionally, SL-4 arises in segment 2, and the Y-1 structure in segment 3 is modified.When describing a genome cyclization model, the region located downstream of the start codon (5 ′ DAR) forms hairpin structures, thereby facilitating the replication initiation step.These regions, when they approach each other, activate viral RNA-dependent RNA polymerase [25].The 5 ′ UTR of all the segments comprises the 5 ′ -GCAC-3 ′ sequence that binds the La protein or its complementary 5 ′ -GUGC-3 ′ sequence, which may facilitate the stabilization of the replication complex [26].Mutations in the 5 ′ -GCAC-3 ′ motif are known to alter the binding affinity of La to the hepatitis C virus IRES and have a profound effect on its translation both in vitro and ex vivo [27].A highly conserved orthoflaviviral 5 ′ -CACAG-3 ′ pentanucleotide was found in hairpin 3 of the Y-1 structure of the segment 1 3 ′ UTR.This region is conserved in segment 2's 3 ′ UTR only in the KITV/2017/1 isolate and is located in hairpin 7 of Y-3.In flaviviruses, this pentanucleotide is located in the 3 ′ long stable hairpin (3 ′ LSH) that is involved in the initiation of genome replication [28].KITV segment 2's 3 ′ UTR is the most heterogeneous.It contains three homologous 5 ′ UTR sequences R1-R3 and two 40-nucleotide repeats.In the KITV/2018/1 and KITV/2018/2 strains, a 35-nucleotide deletion occurs in the second repeat.In KITV segment 2's 3 ′ UTR, two pseudoknots, PK1 and PK2, were found, which are responsible for stabilizing the secondary structure of this region in orthoflaviviruses [29].These results suggest an unusual evolutionary relationship between the unsegmented and segmented viruses of the Flaviviridae family.
Furthermore, multiple UAG sites (two to six) for interactions with the RNA-binding protein Musashi-1 (MSI1) were found in the 3 ′ UTR of all the KITV RNA segments.The data are shown in Table S3.In comparison, African Zika virus and European tick-borne encephalitis virus contain six MSI1 binding sites.In this case, the saturation coefficient of the 3 ′ UTR with the UAG trinucleotides in Zika virus is 1.09, which indicates a high level of binding sites relative to the 3 ′ UTR's size, whereas the same coefficient in tick-borne encephalitis virus is 0.55 [30].According to in vivo studies, MSII enhances virus replication, regulates translation, and is involved in Zika virus-induced neurotropism through interaction with the 3 ′ UTR of Zika virus RNA [31,32].Zika virus RNA may compete with endogenous targets for the binding of MSI1 in the developing embryonic brain, thereby dysregulating the expression of genes essential for neural stem cell development, which suggests the presence of mechanisms of MSI1-mediated congenital neuropathology [33].Therefore, many binding sites of the 3 ′ UTR of KITV to MSI1 may indicate a possible neurotropic potential of this virus, which requires further research to clarify the epidemiological significance of this unusual multicomponent flavi-like virus.
The 5 ′ UTR and 3 ′ UTR of segmented flavi-like viruses have been found to consist of patterns of repeating sequences (conserved motifs).The structure and number of conserved motifs are individual to each virus.However, the 5 ′ UTR and 3 ′ UTR of all the segments contained the 5 ′ -CAAGUG-3 ′ sequence in at least one conserved motif.These data are consistent with a previously published paper on the search for conserved sequences in the untranslated regions of JMVs [12].Analysis of the obtained models revealed that the 5 ′ -CAAGUG-3 ′ sequence often forms the first hairpin of the 5 ′ UTR and hairpins of the 3 ′ UTR Y-1 structure.These regions are shown in Figures S1-S7, respectively.KITV is also characterized by the presence of 5 ′ -CAAGUG-3 ′ in hairpin 1 of the 5 ′ UTR of segments 1, 2, and 4 and in the hairpins of the Y-1 structure of the 3 ′ UTR of segments 2 and 4. As reported, JMTV retained the 5 ′ -CAAGUG-3 ′ sequence in the UTR of all the segments during its first isolation, which indicated that all four segments belonged to the same virus [2].The conserved location of the 5 ′ -CAAGUG-3 ′ sequence in the first structures of the untranslated regions may indicate that this region is involved in interactions with viral and/or cellular proteins to regulate the virus's life cycle, similarly to how it occurs in other viruses of the Orthoflavivirus genus [34][35][36][37][38].

Figure 1 .
Figure 1.Phylogenetic analysis of tick-borne flavi-like viruses with a segmented genome according to the nucleotide sequence ORF of the all segments.(A) Phylogenetic analysis of the ORF nucleotide

Figure 1 .
Figure 1.Phylogenetic analysis of tick-borne flavi-like viruses with a segmented genome according to the nucleotide sequence ORF of the all segments.(A) Phylogenetic analysis of the ORF nucleotide sequence encoding RNA-dependent RNA polymerase.(B) Phylogenetic analysis of the ORF nucleotide sequence encoding VP1.(C) Phylogenetic analysis of the ORF nucleotide sequence encoding the NS3 viral protein.(D) Phylogenetic analysis of the ORF nucleotide sequence encoding VP2.(E) Phylogenetic analysis of the ORF nucleotide sequence encoding VP3.

Figure 2 .
Figure 2. Linear models of the secondary structure of 5′ UTR (a) and 3′ UTR (b) of genomic RNA segment 1 of the KITV isolate KITV/2018/1.Functional orthoflavivirus regions are indicated by colored arrows.

Figure 2 .
Figure 2. Linear models of the secondary structure of 5 ′ UTR (a) and 3 ′ UTR (b) of genomic RNA segment 1 of the KITV isolate KITV/2018/1.Functional orthoflavivirus regions are indicated by colored arrows.

Figure 4 .
Figure 4. Linear model of the secondary structure of the 5′ UTR of genomic RNA segment 3 of the KITV/2018/1 isolate.Functional orthoflavivirus regions are indicated by colored arrows.

Figure 4 .
Figure 4. Linear model of the secondary structure of the 5 ′ UTR of genomic RNA segment 3 of the KITV/2018/1 isolate.Functional orthoflavivirus regions are indicated by colored arrows.

3. 6 .
KITV Segment 4's 5 ′ and 3 ′ UTR Structures The secondary structure of segment 4's 5 ′ UTR is represented by SL-1, SL-2, and SL-3, and that of segment 4's 3 ′ UTR is represented by two Y-structures.Models of secondary structure are shown in Figure 5.The 5 ′ UTR SL-1 structure comprises the La binding region and the 5 ′ -UGGCAAGUGC-3 ′ R1 sequence previously found in KITV segment 2's 5 ′ and 3 ′ UTRs.The same R1 sequence is present in the second hairpin of the 3 ′ UTR Y-1 structure.No other regulatory elements were found.

Figure 5 .
Figure 5. Linear models of the secondary structure of 5′ UTR (a) and 3′ UTR (b) of genomic RNA segment 4 of the KITV isolate KITV/2018/1.Functional orthoflavivirus regions are indicated by colored arrows.

Figure 5 .
Figure 5. Linear models of the secondary structure of 5 ′ UTR (a) and 3 ′ UTR (b) of genomic RNA segment 4 of the KITV isolate KITV/2018/1.Functional orthoflavivirus regions are indicated by colored arrows.

Figure 6 .
Figure 6.Conservative motifs in 5′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 2. (b).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. (c).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 3 of segmented flavi-like viruses.A highly conservative area is highlighted in motifs 1 and 4. (d).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in Figures S8-S11.
Figure 6.Conservative motifs in 5′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 2. (b).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. (c).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 3 of segmented flavi-like viruses.A highly conservative area is highlighted in motifs 1 and 4. (d).Nucleotide sequences of motifs and their location in the 5′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in Figures S8-S11.

Figure 6 .
Figure 6.Conservative motifs in 5 ′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 2. (b).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. (c).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 3 of segmented flavi-like viruses.A highly conservative area is highlighted in motifs 1 and 4. (d).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in Figures S8-S11.
Figure 6.Conservative motifs in 5 ′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 2. (b).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. (c).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 3 of segmented flavi-like viruses.A highly conservative area is highlighted in motifs 1 and 4. (d).Nucleotide sequences of motifs and their location in the 5 ′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 3. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in Figures S8-S11.

Figure 7 .
Figure 7. Conservative motifs in 3′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 3′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (b).Nucleotide sequences of motifs and their location in the 3′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (c).Nucleotide sequences of motifs and their location in the 3′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in the Figures S12-S14.
Figure 7. Conservative motifs in 3′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 3′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (b).Nucleotide sequences of motifs and their location in the 3′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (c).Nucleotide sequences of motifs and their location in the 3′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in the Figures S12-S14.

Figure 7 .
Figure 7. Conservative motifs in 3 ′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 3 ′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (b).Nucleotide sequences of motifs and their location in the 3 ′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (c).Nucleotide sequences of motifs and their location in the 3 ′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in the Figures S12-S14.
Figure 7. Conservative motifs in 3 ′ UTR segmented flavi-like viruses.(a).Nucleotide sequences of motifs and their location in the 3 ′ UTR of segment 1 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (b).Nucleotide sequences of motifs and their location in the 3 ′ UTR of segment 2 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. (c).Nucleotide sequences of motifs and their location in the 3 ′ UTR of segment 4 of segmented flavi-like viruses.A highly conservative area is highlighted in motif 4. The size of the bases correlates with the conservation of their detection among different JMV isolates.Complete date are presented in the Figures S12-S14.