Ethical statement and Sample collection
All animal experiments were performed with approval from the Animal Care and Use Committee of Hokkaido University following the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology in Japan (permit number 19-0019). Adult host-questing ticks were captured using the flagging method, and blood-sucking ticks were collected from domestic animals (e.g., cattle, goat, sheep, and dogs) in Isoka (10.15°S, 32.63°E), Mpulungu (8.76°S, 31.11°E), and Samfya (11.36°S, 29.55° E) from November 2017 to January 2018 in Zambia. Collected ticks (n = 573) were morphologically identified under a stereomicroscope (see Supplementary Table S1 online). Each tick was washed in 70% ethanol containing 1% iodine and then submerged in distilled water. The samples were then homogenized with 200 μl of Dulbecco’s modified Eagle’s medium using a homogenizer (Tomy Seiko, Tokyo, Japan) at 3,000 rpm. Total RNAs were extracted from 100 μl of the homogenates using TRIzol-LS (Invitrogen, Waltham, MA) according to the manufacturer’s protocol, and remaining lysate samples were stored at −80°C until use for virus isolation.
Detection of flavivirus
Tick RNA samples were examined to detect flavivirus via RT-PCR using a One Step RT-PCR Kit v2 (Takara, Shiga, Japan) with pan-flavivirus primer set (see Supplementary Table S2 online) based on the conserved sequence within the flavivirus NS5 protein as previously described53. The RT-PCR conditions were as follows: initial reverse transcription step at 50°C for 30 min; PCR activation step at 94°C for 2 min; 43 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR products were subjected to direct sequencing using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).
Virus isolation
Tick homogenates positive for flavivirus were cultured in Vero E6 (kindly provided by Dr. Heinz Feldmann, National Institutes of Health, Bethesda, MD), BHK-21 (gift from Dr. Akira Oya, the National Institutes of Health, now National Institute of Infectious Diseases, Tokyo, Japan), ISE6 (kindly provided by Dr. Ulrike Munderloh, University of Minnesota, Saint Paul, MN) or C6/36 cells (purchased from the American Type Culture Collection, Manassas, VA). Tick lysates were inoculated into these cell lines, and supernatants and cell lysates were examined at each passage for detection of flavivirus genome by RT-PCR of NS5. Lysates were also inoculated into neonatal mice brain via intracerebral injections, and brain-derived RNA was subsequently subjected to RT-PCR.
Library preparation and whole genome sequencing
Total RNAs extracted from tick homogenates positive for flavivirus were used for whole genome sequencing. Ribosomal RNA depletion from total RNA was performed using RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen), and cDNA was synthesized using a PrimeScript Double Strand cDNA Synthesis Kit (Takara) according to the manufacturers’ instructions. The cDNA libraries were prepared using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions, and were then subjected to whole-genome sequencing on a MiSeq using a MiSeq Reagent Kit v3 (600 cycles) (Illumina). Sequencing data was analyzed using the CLC Genomics Workbench software (CLC bio, Hilden, Germany). Flavivirus genome contigs were obtained by de novo assembly and the overlapped contig sequences were confirmed by PCR amplification with specific primers and Sanger sequencing. The 5′ and 3′ termini of the flavivirus genome were amplified using RACE with specific primers and a SMARTer RACE cDNA Amplification Kit (Takara) according to the manufacturer’s protocol (see Supplementary Table S2 online). Amplified products were directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit.
Genetic comparison and phylogenetic analyses of flaviviruses
Polyprotein ORF positions were predicted using GENETYX version 12 (GENETYX Corporation, Tokyo, Japan). The putative cleavage sites of the detected flavivirus were determined by comparison with known cleavage sites of previously characterized flaviviruses as well as cleavage patterns of a host signal peptidase, furin and viral serine protease as previously described54. Bioinformatic analyses were performed using flavivirus sequences deposited in the DDBJ/EMBL-Bank/GenBank databases. Identity comparison analyses were conducted among flaviviruses using GENETYX version 12. Conserved enzymatic motifs of NS3 and NS5 were identified by sequence alignments of detected and previously characterized flaviviruses. The hydropathy profiles of viral proteins were obtained using the web-based tool ProtScale (https://web.expasy.org/protscale/) from the ExPASy Bioinformatics Resource Portal with the Kyte and Doolittle scale option55. Phylogenetic analyses based on the amino acid sequence of flavivirus polyprotein and each viral protein were performed using MEGA756. The MUSCLE protocol was used to align the sequences, and phylogenetic trees were constructed using the maximum-likelihood method based on the Tamura-Nei model with 1,000 bootstrap replicates.
Compositional analysis
Complete or nearly complete genome sequences from 128 flaviviruses with defined host range and transmissibility were used as a dataset, which were classified as TBFV, MBFV, NKV, ISFV, and flaviviruses derived from marine organisms (i.e., Marine), and used for analyses (see Supplementary Table S4 online). The dinucleotide ratios (observed/expected values) were calculated using the formula PXY=ƒXY/ƒXƒY, in which ƒX and ƒY denote the frequencies of the mononucleotides X and Y, respectively, and ƒXY denotes the frequency of dinucleotide XY42. Linear discriminant analysis was performed using the R package (The R Project for Statistical Computing, 2018).
Prediction of RNA secondary structure
RNA secondary structure predictions in both MPFV UTRs were computed using RNAfold of the Vienna RNA Package57, explicitly disallowing isolated base pairs (--noLP option). Structural homology of the predicted 5′-UTR stem-loop structures SLA and SLB to these elements in other flaviviruses was determined by Infernal covariance models (CMs)58. Likewise, TBFV-specific CMs from a recent study30 were used to confirm the predicted locus of the MPLV 3′SL element, and the Rfam59 CM RF00525 (Flavivirus_DB) was used to annotate the single DB element in the 3′-UTR. All secondary structure plots were produced with the RNAplot utility57.
Design of leader sequences for the Xrn1 degradation assay
To test the capacity of the predicted 3′-UTR structures xrRNA1 and xrRNA2 to inhibit nuclease digestion, we performed an Xrn1 degradation assay according to the methods of Chapman et al.23. To this end, we extracted the nucleotide sequences folding into three-way junction structures together with their downstream hairpins. The degradation assay requires a leader sequence upstream of the xrRNA to load Xrn1. To exclude the possibility that the leader sequence interacts with the sequence forming the three-way junction, we designed custom, artificial 31 nt leader sequences that do not form any significant secondary structures nor distort the canonical xrRNA fold. We sampled sequences fulfilling this requirement using RNAblueprint46. We employed partition function folding with default parameters implemented in the ViennaRNA package to obtain ensemble free energies ΔG, optimizing for a maximal (ZF/Z) ratio as design goal, where ZF is the partition function under the constraint that the three-way junction and downstream hairpin are formed in the presence of the designed 31 nt leader sequence, and Z is the unconstrained partition function. Z is related to the ensemble free energy via ΔG=-RTlnZ, where R is the universal gas constant and T is the thermodynamic temperature. Reliability of the predicted structures, including the designed leader sequences was, visualized in terms of positional entropy (Figs. 6a and 6b).
Xrn1 degradation assay
Two 3'-UTR RNAs [(+31)-xrRNA1 and (+31)-xrRNA2] were chemically synthesized and purified by the Agilent 1290 Infinity II chromatography system (Agilent Technologies, Santa Clara, CA; see Supplementary Table S5 online). The synthesized RNAs were phosphorylated using T4 Polynucleotide Kinase (Takara) and purified by the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The modified RNAs were incubated at 90°C for 2 min, followed by 20°C for 5 min, then held at 4°C for RNA folding. Xrn1 digestion reactions were conducted with 3–4 µg of the 3'-UTR RNA (~ 100 pmol) and 2 µg of 5' monophosphorylated 31-mer control RNA (~ 200 pmol) in NEB3 buffer [100 mM NaCl, 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, and 1 mM DTT] (New England Biolabs, Ipswich, MA). The RNA mixture was split between two tubes, and two units of Xrn1 (New England Biolabs) were added to one aliquot while the other served as an Xrn1-negative control. Both mixtures were incubated at 37°C for 2 h, followed by quenching via addition of an equal volume of Novex TBE-Urea Sample Buffer (Invitrogen). The RNA products were analyzed on 15% denaturing PAGE gel and visualized by ethidium bromide staining.
Mapping Xrn1 halt sites of 3'-UTR RNAs
Xrn1 halt sites were mapped by RNA sequencing utilizing the primer extension method60. RNA products remaining after the Xrn1 degradation assay were recovered using the ZR small-RNA PAGE Recovery Kit (ZYMO research, Irvine, CA) according to the manufacturer’s instruction. Reverse transcription was carried out using SuperScript IV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instruction. Briefly, approximately 2 pmol of recovered RNA products were annealed to the 5′ end-labeled primer with deoxyribonucleoside triphosphates and dideoxynucleoside triphosphates (see Supplementary Table S2 online). Reverse transcriptase and buffer components were added to the primer-RNA hybrids to catalyze elongation of the primer to 5′ end of the RNA. Following the elongation reaction, equal volumes of loading buffer (95% formamide and 10 mM EDTA) were added to the reactions, and the mixtures were then incubated at 80°C for 5 min. The resulting fluorescein-labeled cDNA products were analyzed on a 15% denaturing PAGE gel and visualized by the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA).
Data Availability Statement
The sequence of Mpulungu flavivirus was deposited into the GenBank/EMBL/DDBJ database (Accession No. LC582740). All data generated or analyzed within this study are included in this published manuscript and its supplementary information files.