The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution. Host proteins are identified which are required to reconstitute a functional RNA phage MS2 replication machinery, providing a promising platform for cell-free gene expression. Due to its simple architecture, MS2 finds extensive use as a model organism in various biological research fields and helped to reveal fundamental biological processes such as translational control, feedback inhibition and key aspects of viral life cycles6. Moreover, MS2 is used as a surrogate for human pathogenic RNA viruses to study the properties, stability and detectability of RNA viruses under different environmental conditions9. Apart from their relevance to RNA biochemistry and molecular biology, protein and RNA components of ssRNA coliphages such as Qβ and MS2 serve as attractive platforms for RNA imaging12, RNA packaging and delivery15 and molecular evolution and gene expression in cell-free systems20.EEscherichia coli cells by MS2 is dependent on the maturation protein (mp), which is present as a single copy in the MS2 capsid and enables binding of MS2 virions to the host’s F-pili6. Following infection, the host translation machinery immediately begins synthesising the MS2 coat protein (cp), whose main function is to encapsulate newly synthesised viral (+) strands. Ribosomal readthrough events of this gene provide a brief opportunity for initiation of translation of the replicase (rep) gene, whose ribosome-binding site is otherwise sequestered in an “operator” hairpin RNA structure21 and inhibited by long-range RNA-RNA interactions22. Similarly, timing and level of mp expression are controlled by the RNA folding kinetics of an untranslated leader sequence23. To initiate genome replication, the MS2rep subunit forms an active holocomplex, which likely consists of the E. coli elongation factors EF-Tu, EF-Ts, ribosomal protein S1 and a yet undefined host protein  strand using antigenomic (−) strand intermediates as template. At later stages of the life cycle, cp dimers bind and stabilise the operator hairpin which ultimately leads to a complete suppression of translation of the rep gene21. Finally, expression of the lysis protein (lys) leads to the release of mature virions from the infected host, enabling new cycles of infection6.Infection of male 24, and are therefore attractive model systems for drug discovery and mutational analyses. However, although MS2 is one of the main model systems for RNA viruses, fundamental aspects of its genome replication remain unclear to date. In particular, a host factor postulated almost 50 years ago26, to be required for RNA replicase activity, remains unknown. Here, using our previously established reporter system for MS2 replicase activity in PURE systems20, we are able to characterise several host factors that modulate the activity of the phage enzyme. Through stepwise reduction of PURE proteins, we identified initiation factor 1 (IF1) as a co-factor that stimulates the activity of the MS2 replicase core complex consisting of MS2rep, EF-Tu, EF-Ts and ribosomal protein S1. Furthermore, we found that initiation factor 3 (IF3) acts as an inhibitor for replicase activity, which has potential implications for the viral life cycle. Finally, we demonstrate that the MS2 replicase complex can evolve small replicable RNA species during the in vitro replication of the full-length genome. These new RNA scaffolds may be well-suited for the generation of robust self-replicating synthetic RNA systems in cell-free in vitro systems.Emesviral replicases are amongst the most ancient RNA-dependent polymerase enzymes and share key characteristics with other viral RNA polymerasesE. coli and purified it by single-step immobilised metal ion affinity chromatography. In the one-step purification protocol, MS2rep co-eluted with the two potential host factors ribosomal protein S1 and the translation factor (TF) EF-Ts, both of which are components of the related Qβ replicase complex  and Emesvirus (MS2) show unexpected differences.To characterise the activity requirements of the MS2 replicase complex, we overexpressed the MS2rep subunit in 27. In this assay, fluorescence from the fluorogenic ligand DFHBI-1T is observed upon (+) strand synthesis of the DFHBI-1T-binding broccoli aptamer28 from its reverse complement, which has been embedded between the reverse complement of both untranslated regions of the MS2 genome ([F30-Bro(−)]UTR(−)) ]UTR(−) and MS2·S1·EF-Ts were incubated in the commercially available PURExpress® system. We also observed aptamer synthesis, albeit at a lower level, in an in-house PURE system (PURE 3.0), which was prepared according to a previously established protocol29  systems−)) Fig. . In agrel29 Fig. . Based oE. coli proteins that are required for MS2 replicase activity, we investigated the activity in the presence of different PURE protein fractions. In PURE 3.0, 30 of the 31 E. coli TFs are obtained after co-expression and purification of the TF genes from three large expression plasmids resulting in three protein fractions 29. An additional enzyme mix contains 70S ribosomes as well as the elongation factor EF-Tu and the enzymes necessary to reconstitute an NTP regeneration system based on creatine phosphate ]UTR(+) in the PUREred environment  synthesis  synthesis upon omission of the individual LD protein fractions. Omission of both LD1 and LD3 had a modest enhancing effect on MS2rep activity which may not even be significant. This is likely caused by the altered crowding conditions in the absence of the protein factors, which may affect RNA folding and/or the formation of inert duplexes32. In contrast, omission of LD2 caused a drastic loss of [F30-Bro(+)]UTR(+) transcription activity , the translation elongation factor EF-Ts (which co-purifies with the MS2rep subunit), and the two translation initiation factors IF1 and IF329. To identify TFs responsible for this marked effect on replicase activity, we performed selective depletion experiments with all LD2 proteins  template. We further observed an unexpected influence on transcription activity by the initiation factors IF1 and IF3. While omission of IF1 caused a ~50% reduction in [F30-Bro(+)]UTR(+) synthesis, the absence of IF3 led to a >400% increase in transcription activity compared to the positive control containing the full 1x LD2 protein fraction. We excluded the possibility that the increase in fluorescence was a result from a direct interaction of the initiation factors with the aptamer domain of [F30-Bro(+)]UTR(+) since neither protein affected the fluorescence intensity of pre-synthesised [F30-Bro(+)]UTR(+) in separate control experiments  synthesis. In contrast, the omission or supplementation of the four tRNA synthetases had no impact on MS2 replicase activity under these conditions. Both the inhibitory effect of IF3 and stimulating effect of IF1 showed clear dose dependence with observable effects even at low micromolar concentrations ]ity Fig.  suggestiins Fig. . Omittinons Fig. , supportons Fig. .Fig. 3UTR(+) synthesis as a function of [F30-Bro(−)]UTR(−) input concentration in the presence of IF3. As expected, the addition of micomolar amounts of IF3 raised the apparent half-saturation concentration of MS2rep for the RNA template while not affecting the maximum rate of transcription at saturating [F30-Bro(−)]UTR(−) concentrations , which encodes the sense strand of the broccoli aptamer, and additional electrophoretic mobility shift assays (EMSAs), we determined an apparent dissociation constant KD of ~3.5 µM for the interaction between IF3 and [F30-Bro(+/−)]UTR(−). This affinity correlates well with the dose dependence observed for transcription inhibition of MS2rep by IF3 ]UTR(−), which is not surprising given the low molecular weight of IF1 (9.1 kDa) in relation to the RNA template (132 kDa). In EMSA experiments based on a Tris/acetate buffer system, the RNA bands showed only a mild smearing behaviour at increasing IF1 concentrations  synthesis enables monitoring (+) strand synthesis from an artificial template, it provides no information on the complete replication cycle of the natural ~3600 nt MS2 genome. To probe full-length genome replication by the in vitro reconstituted MS2 replicase complex, we integrated the broccoli aptamer into the (+) strand of MS2 wild-type (MS2wt) genome at an amenable site downstream of the open reading frame of the maturation protein ]MS2(+), we observed a continuous increase in DFHBI-1T fluorescence when the MS2rep·S1·EF-Ts complex was incubated in the PURExpress system, which suggests processive genome replication  showed an estimated sixfold amplification, corresponding to an increase of [F30-Bro(+)]MS2(+) from 15 nM to ~90 nM over 4 h.While [F30-Bro(+)]ein Fig. , where iion Fig. . CompariMS2(+/−) templates produced only a weak fluorescence output compared to synthesis of [F30-Bro(+)]UTR(+) using [F30-Bro(−)]UTR(−) as template  construct used during initial experiments  and (−) strands Fig. . Synthesate Fig. . Notablynts Fig. . This fiMS2(+) from [F30-Bro(−)]MS2(−) template in the presence of IF1  strand synthesis was not affected by supplementation of IF1 Fig. . However43. In contrast, replication of the full-length genome in PURE systems enabled sustained synthesis of the genomic (+) strand. Under these more in vivo-like conditions, ribosome binding and translation to the MS2(+) RNA may counteract direct annealing with newly synthesised (−) strand, thereby supporting continuous RNA replication similar to what has been described for in vitro replication by the Qβ replicase complex44.In conclusion, the isolated MS2 replicase holocomplex was able to synthesise genomic RNA from both (+) and (−) strand templates in batch reactions. However, under these conditions, both strands readily anneal to form double-stranded RNA, which can no longer be used as a template for replication47. To test if MS2rep is capable of a similar spontaneous generation of short amplifiable RNA species, we compared the activity of both purified Qβ and reconstituted MS2 core complex in template-free reactions supplemented with NTPs and SYBR Green nucleic acid stain. As expected, we observed a rapid increase in fluorescence after a brief lag phase of ~5–10 min in the presence of the Qβ heterotetramer  RNA in vitro49, the MS2 replicase complex shows a number of different characteristics.Previous attempts to characterise emesviral replicases in vitro were hampered by the general lability of the enzymes and the dependency of replicase activity on an unknown, yet easily dissociable, host factorFirstly, EF-Tu, which is an integral part of the active complex for both replicases, only weakly binds to the stable MS2rep·S1·EF-Ts core trimer. Secondly, we identified IF1 as the previously unknown host factor improving MS2rep activity in vitro.37. Indeed, IF1 acts as a transcription anti-termination factor, which can destabilise strong secondary structure elements and thereby facilitating RNA polymerase readthrough38. Furthermore, IF1 showed RNA chaperone activity during trans-splicing assays both in vivo and in vitro36. These properties and our own findings suggest that IF1 supports MS2 genome replication by destabilising secondary structure elements of single-stranded templates and exposing terminal and internal binding sites for the MS2 replicase and/or through facilitating replication initiation and product release. The influence of IF1 on RNA synthesis resembles the role of the OB proteins S1 and Hfq for (−) strand synthesis during the Qβ replication cycle. Here, Hfq facilitates the access of replicase to the 3′ end of the genomic (+) strand51, while S1 appears to contribute to both termination of replication and re-initiation after product release52. Thus, a similar yet MS2-specific role of IF1 for the remodelling of MS2 templates seems plausible.IF1 is a S1 domain protein that contains an oligomer binding (OB) fold found in a variety of RNA chaperones35, which we reproduced and quantified in our study. Furthermore, our results suggest a direct competition for the 3′ end between the replicase and IF3 because increasing concentrations of either the replicase or the substrate RNA compensated for the inhibitory effect of IF3. Notably, MS2 gene expression was also shown to be completely independent from IF3, unlike for E. coli host proteins53. Thus, replication and translation of MS2 genomes seem to be well adapted to conditions under which IF3 levels are minimal, such as it was reported for E. coli cells that have reached stationary phase54. While phage virions are still produced under these conditions, protein synthesis rates are usually insufficient to trigger cell lysis and phage release55. Therefore, we speculate that the ability of the MS2 replication system to continuously produce infectious but host-contained virions in slowly growing cells may enable phage persistence within starved bacterial populations. This state may last until growth conditions and therefore a lytic reproduction cycle can be restored. However, in initial experiments under laboratory growth conditions, we did not observe any noteworthy effect on MS2 infectivity upon overexpression of the initiation factors in a laboratory E. coli F+ strain. Neither the number nor the morphology of plaques were largely altered upon over-expression of IF1, IF3, and, as a control AsnRS  E. coli cells, respectively, (Thermo Fisher Scientific) were transformed by electroporation with either pBAD33-based (Top10) or pLD1-3 plasmids (BL21(DE3)). Proteins were overexpressed at 16 °C overnight in Lysogeny Broth (LB Lennox) with 0.2% L-Arabinose for all pBAD33-based constructs or 1 mM IPTG for pLD1-3, respectively. Subsequently, proteins were purified over HisPur™ Ni-NTA resin (Thermo Fisher Scientific) using HEPES buffer  and stored in HEPES/glycerol buffer . Preparations from pLD1, pLD2 and pLD3 were adjusted to ten-fold stocks with the final protein content per reaction equalling concentrations as described previously29. A more detailed protocol is included in the The preparation of proteins followed an adapted version of the protocol described by Shepherd et al.20. For experiments using solution A  and B (containing ribosomes and enzymes) of the PURExpress® In Vitro Protein Synthesis Kit (NEB), standard reactions were supplemented with 10 µM DFHBI-1T ((Z)-4--2-methyl-1--1H-imidazol-5(4H)-one))28, MS2rep and [F30-Bro(−)]UTR(−) or [F30-Bro(+/−)]MS2(+/−), respectively, as described for the individual experiments:All readout constructs are based on designs by Weise et al.UTR(−) and 10 µM DFHBI-1T300 nM MS2rep, 150 nM [F30-Bro(−)]MS2(−) and 10 µM DFHBI-1T1 µM MS2rep, 15 nM [F30-Bro(−)]All experiments based on the in-house PURE system (PURE 3.0/ PUREred) were assembled including 0.5 mM each ATP, GTP, CTP and UTP, 10 mM DTT, 15 µM EF-Tu, 1.5 µM S1 and all other components as follows.UTR(−), 1x LD1, LD2 and LD3 and 10 µM DFHBI-1T300 nM MS2rep, 150 nM [F30-Bro(−)]UTR(−), 5 µM LD2 components and 10 µM DFHBI-1T300 nM MS2rep, 50 nM [F30-Bro(−)]UTR(−), 15 µM EF-Ts, 5 µM LD2 components and 10 µM DFHBI-1T300 nM MS2rep, 50 nM [F30-Bro(−)]UTR(−), 15 µM EF-Ts and 10 µM DFHBI-1T300 nM MS2rep, 50 nM [F30-Bro(−)]MS2(+/−), 15 µM EF-Ts, 10 mM DFHBI-1T and 0.5 U per µL RNase inhibitor (moloX)1 µM nM MS2rep, 50 nM [F30-Bro(−)]2 was 6 mM, with HEPES, KCl, and glycerol supplemented in 50 mM, 100 mM, and 18%, respectively. All reactions were prepared in MicroAmp Fast 8-TubeStrips (Thermo Fisher Scientific) and incubated at 37 °C in a StepOne Real-Time PCR System (Thermo Fisher Scientific). Fluorescence signals were recorded every 60 s over a total of four hours or every 120 s for six hours for experiments involving [F30-Bro(+/−)]UTR(+/−) or [F30-Bro(+/−)]MS2(+/−), respectively. Unless stated otherwise, all experiments were performed in independent triplicates assembled from the same stock solutions.The final concentration of MgClE. coli Poly(A) Polymerase (NEB) and re-purified with the same clean-up kit. Double-stranded cDNAs were synthesised using Template Switching RT Enzyme Mix (NEB) in combination with primers CDSII-T24VN and TSO-CDSII (Supplementary Table nd Strand cDNA Synthesis protocol. DNAs were purified using Monarch® PCR & DNA Cleanup Kit (NEB) and subsequently prepared for cloning with NEB® PCR Cloning Kit (NEB). For transformation, 2 µL of the reactions were transformed into chemically competent Top10 E. coli cells (Thermo Fisher Scientific).To obtain sequencing data, RNAs from serial transfer experiments see  were pur2 was 6 mM, with HEPES, KCl, and glycerol supplemented at 50 mM, 100 mM, and 18%, respectively. MS2rep was supplemented at 500 nM and RNAs in the concentrations described in Supplementary Fig. The replication capability of MSRP-22 was analysed by gel electrophoresis. The samples for the replication time course were obtained by preparing the following reaction mixtures and incubating at 37 °C: 15 µM EF-Tu, 15 µM EF-Ts, 1.5 µM S1, 0.5 mM of each of ATP, GTP, CTP and UTP, and 10 mM DTT. The final concentration of MgClAll samples for gel electrophoresis were prepared by mixing equal volumes of reaction mixture and 2x RNA Gel loading dye (Thermo Fisher Scientific). Samples were heated to 70 °C for 5 min and then slowly cooled to room temperature. This allowed bound proteins to denature and dissociate from RNAs, as well as the complementary RNA strands to denature and anneal.The methods for statistical analysis and sizes of the samples (defined as n) are specified in both the results section and individual figure legends for all of the quantitative data. All values are presented as mean ± SD with the indicated sample size where applicable.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Anopheles spp-asscoaited viruses, and of the dual-host associated ISF Binjari virus contain duplicated xrRNAs. We also reveal novel structural elements in the 3’UTRs of dual host-associated and Anopheles-associated classical ISFs. Structure-based phylogenetic analysis demonstrates that xrRNAs identified in Anopheles spp-associated ISF are likely ancestral to xrRNAs of ISFs and MBFs. In addition, our data provide evidence that duplicated xrRNAs are selected in the evolution of flaviviruses to provide functional redundancy, which preserves the production of sfRNAs if one of the structures is disabled by mutations or misfolding.Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. To date, the ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. Herein the production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3’UTRs are determined. The xrRNAs responsible for the biogenesis of ISF sfRNAs are also identified, and the role of these sfRNAs in virus replication is demonstrated. We demonstrate that 3’UTRs of all classical ISFs, except  Subgenomic flaviviral RNAs (sfRNAs) are produced by pathogenic flaviviruses to counteract the host antiviral response. sfRNAs are products of incomplete degradation of viral RNA by the host exoribonuclease XRN1 which stalls on XRN1-resistant structural elements (xrRNAs) in the 3‟UTR. Here, Slonchak et al. identify xrRNAs for mosquito-specific flaviviruses, characterize their ability to stall XRN1 and produce sfRNAs, and apply structure-based phylogenetic analysis to provide evidence for evolutionary selection of these elements. Flavivirus genus can be divided into the following ecological groups: mosquito-borne flaviviruses (MBFs), which circulate between mosquito and vertebrate  hosts; tick-borne flaviviruses (TBFs) that are maintained in nature in tick-vertebrate cycle1; viruses that only infect vertebrates and are thought to be transmitted horizontally2  and insect-specific flaviviruses (ISFs) that infect mosquitoes or sand flies and are believed to circulate predominantly via vertical transmission3. To date, pathogenic dual-host viruses (mosquito- and tick-borne flaviviruses) are the most well-studied flaviviruses. To replicate in diverse hosts, these viruses have evolved multiple mechanisms to counteract, subvert and evade antiviral responses of arthropods and vertebrates. One of them is by producing noncoding viral RNA named subgenomic flaviviral RNA (sfRNA) (reviewed in4). In infected cells, viral genomic RNA is subjected to degradation by the host 5’-3’ exoribonuclease XRN1. XRN1 is a highly processive enzyme with a helicase activity, which can unwind and fully digest virtually any RNA5. However, flaviviruses contain uniquely folded RNA elements in their 3’UTRs that can halt the progression of XRN16. Stalling of XRN1 prevents complete degradation of viral genomic RNA and results in accumulation of 3’UTR-derived sfRNAs in the infected cells7  that contain a three-way junction between three RNA helices  and a pseudoknot (PK) formed by the terminal loop (L2) of P2 helix 21. In addition, crystal structures have been solved for three mosquito-borne flavivirus xrRNAs17 and xrRNA of no known vector flavivirus Tamana Bat virus (TABV)22. Based on the conserved structural elements, xrRNAs are classified into two classes – class 1 xrRNAs present in mosquito-borne flaviviruses and class 2 xrRNAs present in tick-borne and no known vector viruses21. Within class 1 xrRNAs, two additional subclasses can be distinguished – subclass 1a includes the typical mosquito borne flavivirus xrRNAs that contain 5-nt P1 helix, 6-nt pseudoknot and conserved unpaired nucleotide between P2 and P316  accumulates mutations in the individual xrRNAs and switches between the production of longer and shorter sfRNAs when switching hosts10. This suggests that duplication of the structural elements within 3’UTR represents an adaptation to the dual host life cycle, and individual sfRNA species are specifically adapted to function in different hosts. However, the apparent functional specialization of sfRNA isoforms was only shown for DENV2, and the hypothesis about structure duplication being an adaptation to host switching has not been unambiguously proven or disproven.3’UTRs of mosquito borne flaviviruses typically contain duplicated stem-loop (SL) based xrRNAs followed by two dumbbell (DB) structures and a 3’-terminal stem-loop (3’SL)21.Despite the extensive knowledge about sfRNAs accumulated to date, several questions regarding their biogenesis, functions, and role in flavivirus evolution still remain unanswered. In particular, it is unknown how the structural diversity of XRN1-resistant elements have evolved and whether the different types of xrRNAs appeared independently in different flaviviruses or diverged from a common ancestor. It is also unclear whether duplicated xrRNAs are unique to mosquito born flaviviruses or present in the viruses with a single-host restricted life cycle. In addition, sfRNAs have been primarily studied in pathogenic flaviviruses, while their biogenesis and functions in non-pathogenic flaviviruses remain largely unknown. For instance, the production of sfRNA has so far been shown only for one insect flavivirus – cell fusing agent virus (CFAV)27. Identifying xrRNA structures responsible for the production of sfRNAs in representatives of both ISF lineages and demonstrating the functional significance of ISF sfRNAs should allow the elucidation of whether production of sfRNAs contributes to host adaptation and unravel how XRN1 resistance has evolved in flaviviruses. Two previous attempts to predict the structure of cISF 3’UTRs did not reveal high-order secondary structures, identifying only short inverted repeats28. However, more recent work suggested that dISFs may contain class 1b xrRNAs, similar to those identified in TBAV23. While the 3’UTRs of dISF were predicted to contain a single SL and a single dumbbell followed by 3’SL24, the role of these predicted structures in XRN1 resistance and production of sfRNAs by dISFs have not been experimentally demonstrated.Herein we aimed to elucidate the biogenesis and functions of sfRNAs in insect-specific flaviviruses. ISFs include two phylogenetically distinct lineages – (i) lineage I or classical ISFs (cISFs) that independently diverged from an ancient flavivirus ancestor and (ii) lineage II or dual host-associated ISFs (dISFs) that are believed to evolve from mosquito-borne flaviviruses that lost the ability to infect vertebratesAnopheles-associated flaviviruses, generate sfRNAs by employing XRN1-resistant structured RNA elements. Using chemical probing for RNA structure, we experimentally determine the secondary structures of the complete 3’UTRs of classical and dual host-associated ISFs. We discover that dISFs contain a novel pseudoknot element that increases XRN1-resistance of class 1a xrRNAs for the robust production of sfRNAs. We also find that cISFs and the dISF BinJV contain multiple copies of xrRNAs. However, Anopheles-associated cISFs contain only a single XRN1-resistant structure, which has features of both class 1a and class 1b structures and is therefore ancestral to xrRNAs of insect-specific and mosquito-borne flaviviruses. Furthermore, our sequence- and structure-based phylogenetic analysis of 3’UTRs reveals that the evolution of flaviviruses involves several xrRNA duplication events that occur independently in cISF, MBF and dISF clades, indicating their significant benefit for viral fitness. Finally, we use ISF mutants with impaired functions of individual or all xrRNAs to demonstrate that different sfRNA species likely have redundant functions in ISFs. Our data thus indicate that duplication of xrRNAs and the acquisition of novel structural elements that increase XRN1-resistance of xrRNAs is an evolutionarily conserved mechanism to ensure robust sfRNA production and protection from potentially damaging impacts of inadvertent mutations.Herein we demonstrate that dISFs and cISFs, including the most phylogenetically distant Aedes-associated cISF Parramatta River Virus (PaRV)29, Culex-associated cISF Palm Creek virus (PCV)30 and dISFs Binjari virus (BinJV)32 and Hidden valley virus (HVV)32. To test for production of sfRNA by Anopheles-associated Karumba virus (KRBV), which is unable to replicate in all tested laboratory cell lines33, RNA from virus-positive field-collected mosquitoes was used. All tested viruses were found to produce sfRNAs . We were able to identify 5’-ends of PaRV, PCV and BinJV sfRNAs and map them to viral 3’UTRs .34. The base pairing information is then used to guide the computational folding of RNA structures. SHAPE results demonstrated that the BinJV 3’UTR contains two pseudoknot-forming stem-loops with 3-way junctions, a single copy of dumbbell element and a canonical 3’SL with a preceding short hairpin . This method identifies paired and unpaired nucleotides in the in vitro transcribed and refolded RNApin Fig. . PK-formpin Fig. . Therefopin Fig. . Lookingpin Fig. , we expepin Fig. , and thepin Fig. . Therefoure Fig.  means thure Fig. . In addiure Fig. . This di19 and have not been predicted to form PKs. Interestingly, in contrast to other flaviviruses21, disruption of PK1 in xrRNA1 or PK2 in XRNA2 of BinJV by mutations did not completely abolish XRN1-resistance  are unique for BinJV or represent a more common feature for all dISFs, we first used IPknoture Fig. . The covure Fig. . The smaure Fig. , confirmure Fig.  generateTherefore, we showed that dISFs have a novel sub-class of xrRNAs containing additional structural elements formed by the small stem-loop and pseudoknot downstream of the canonical xrRNA structure. In the current classification of xrRNA types, they would be class 1c xrRNAs. They occur commonly in dISFs, but not in MBFs and employ novel pseudoknot interactions to confer an additional XRN1 resistance to the upstream canonical xrRNAs for the production of sfRNAs. In addition, the experimental identification of the secondary structure of dISF 3’UTR demonstrated that this group of flaviviruses can contain duplicated SLs.23 with minor differences. Two of them (SLI and SLIII) were mapped to the 5’-termini of the PaRV sfRNA-1 and sfRNA-3  that partially lost its XRN1 resistance due to a mutation. We also obtained the evidence that multiple xrRNAs in the 3’UTRs of PaRV and PCV emerged due to duplication, which happened early enough in their evolution to allow accumulation of sequence difference between xrRNAs of each virus while maintaining structural conservancy.Anopheles-associated classical ISFs are the most phylogenetically divergent group of insect-specific flaviviruses that were suggested to represent the closest clade to the common ancestors of ISFs and MBFs33. In this study, we used Karumba virus (KRBV) to represent this clade and showed that this virus appears to be the only flavivirus analyzed so far that produced only one sfRNA species , we first performed sequence alignment  for all three clades of flaviviruses to identify their consensus structures. The 3’UTR alignment demonstrated the ancestral position of the des Fig. . The topdes Fig. . This innce Fig. . This fuNAs Fig.  and B inNAs Fig. . This alNAs Fig. . MoreoveAnopheles-associated ISFs. The evolution of insect-specific flaviviruses indicates a strong selective pressure for increasing the cumulative XRN1 resistance in the 3’UTRs either via duplication of existing xrRNAs  or by gaining additional pseudoknots that improve the performance of the existing XRN1-resistant structures. The fact that duplication of xrRNAs independently occurred several times in flavivirus evolution and was further selected indicates that multiple sfRNA species confer an advantage in viral fitness irrespective of which hosts they replicate.The results of phylogenetic analysis collectively indicate that classical insect-specific flaviviruses and mosquito-borne flaviviruses diverged from a common ancestor similar to 10. One of these sfRNAs (shorter) was beneficial for virus replication in the insect host, and another sfRNA (longer) for replication in vertebrates10. Switching between the production of longer and shorter sfRNAs was suggested as part of adaptation for virus shuttling between mosquitoes and vertebrates24. Here we demonstrated that single host (insect-specific) flaviviruses also contain duplicated xrRNAs and produce multiple sfRNA species. As ISFs don’t switch hosts, the duplications of xrRNAs in these viruses should have a different biological function. It is also unclear whether different sfRNA species produced by ISFs are functionally redundant or divergent.A previous study on Dengue viruses proposed that different xrRNAs within the same 3’UTR produced functionally divergent sfRNA speciesTo clarify this matter, we generated mutant ISFs deficient in the production of sfRNAs and assessed their replication. If sfRNA isoforms are redundant, we expected that viruses would tolerate deficiency in single sfRNAs but become attenuated when production of all sfRNAs is impaired. If individual sfRNA species have unique, specialised functions, we expected that loss of each sfRNA should impair virus replication. Therefore, we introduced mutations that prevent PK formation by SLI, SLII and SLIII of PaRV and PCV generating mutants in PK1’, PK2’ and PK3’, respectively. We also generated combined PK1’2’3’ mutants for PaRV and PCV in which all three PKs were mutated. As BinJV contains only two canonical xrRNAs, we generated PK1’, PK2’ and PK1’2’ mutants for this virus. The mutations were the same as those analysed in in vitro XRN1 resistance assays.3 FFU/mL vs ~106-7 FFU/mL observed for other mutants and WT virus). Moreover, sequencing of the recovered virus revealed reversion of mutations in xrRNA1 and xrRNA2 that restored the production of sfRNA-1 and sfRNA-2, respectively  did not affect virus replication Fig. . HoweverCollectively, our results indicate that sfRNA species of different lengths are likely to be functionally redundant in ISFs. Therefore, we propose that duplication of xrRNAs ensures sfRNA production even if one of the structures loses XRN1 resistance. We also showed that in dISF, a similar backup mechanism for sfRNA production is provided by novel pseudoknots located downstream of the canonical xrRNAs. The presence of these novel pseudoknots, which is unique for dISFs, explains why most dISFs tolerated the loss of duplicated xrRNAs without compromising the robustness of sfRNA production and virus viability.4. Herein we demonstrated that classical and dual host-associated insect-specific flaviviruses also produce sfRNAs by employing the XRN1-resistance mechanism. Using RNA SHAPE we, for the first time, determined the secondary structures of complete 3’UTRs in the divergent representatives of cISF and dISF lineages and experimentally identified structured RNA elements that enable XRN1 resistance and production of sfRNAs in these viruses. Conservation of sfRNA production in the highly divergent classical ISFs, dual host-associated ISF BinJV and Anopheles-associated cISF KRBV indicates the crucial importance of sfRNAs for flavivirus replication in insect hosts. Moreover, considering that Anopheles-associated ISFs are believed to be the most closely related viruses to the common flaviviral ancestor33, the production of sfRNA by KRBV indicates that the ability to resist RNA degradation by XRN1 and generate sfRNAs was fixed very early in flavivirus evolution. This further emphasises the importance of sfRNAs in the life cycle of flaviviruses.All dual host flaviviruses tested to date were shown to produce sfRNAs by employing XRN1-resistant structured RNA elements4, while ISFs were predicted to lack these duplications28. In addition, a previous study demonstrated that adaptation of DENV to insect and vertebrate hosts involves switching between the production of shorter and longer sfRNA species, respectively, due to the accumulation of point mutations in corresponding xrRNAs37. Longer sfRNA species benefited DENV replication in vertebrates, while shorter sfRNAs were shown to function in mosquito cells10. It was, therefore, suggested that duplication of xrRNAs occurred in dual host infecting flaviviruses to enable switching between the production of functionally distinct sfRNAs while adapting to replication in different hosts. Based on these observations, the lack of xrRNA duplications in ISFs was proposed as a reason for their host restriction.Most mosquito-borne vertebrate infecting flaviviruses are known to produce at least two sfRNA species due to the presence of duplicated xrRNAs in their 3’UTRs20 and the DENV438 virus contain only one xrRNA while replicating in both insect and vertebrate hosts. Collectively, published data and our results herein indicate that duplication of xrRNAs has not resulted from adaptation to the dual host life cycle and that lack of these duplications is not the mechanism of host restriction in ISFs. Furthermore, we found that deficiency in the production of individual sfRNAs can be tolerated by ISFs, while the deficiency in all sfRNAs is largely detrimental to ISF replication fitness. These results are consistent with previous observations in MBFs. In particular, Zika virus and West Nile virus mutants deficient in single sfRNAs replicated in mosquito and vertebrate cells at the levels comparable to WT virus, whereas mutations that abolished production of both sfRNAs either resulted in a substantial attenuation of virus replication or were lethal40. Therefore, we concluded that in many flaviviruses, including ISFs, different sfRNA species are functionally redundant and that host specialisation of sfRNAs observed for DENV1-3 viruses37 is likely unique to these members of DENV serogroup.Contrary to this, we found that all ISFs analysed in our study except KRBV contained duplicated xrRNAs and produced multiple sfRNAs, although none of the viruses could infect vertebrate cells. Moreover, representatives of the YFV-groupAnopheles-associated cISF KRBV. Together with the structural data for the 3’UTR of KRBV and other ISFs generated here, it enabled us to reconstruct the most complete to date structure-based phylogenetic trees of flavivirus xrRNAs and 3’UTRs and mapping of xrRNA duplication events on them. The Anopheles-associated ISFs contain a single copy of the XRN1-resistant element, which has a general topology of class 1b xrRNA (present in TABV and cISFs) while lacking noncanonical C-A pairing and forming 5-nt pseudoknot, which only occurs in class 1a xrRNAs of MBFs and dISFs. This structure was found to be ancestral for both cISFs and MBFs/dISFs xrRNAs and likely has the same organisation as xrRNA of a common flavivirus ancestor. Notably, viruses from YFV serogroup were most closely related to Anopheles-associated viruses on the MBF branch of the phylogenetic tree  that have homologous sequence and structure. However, only two (SLI-PK1 and SLIII-PK3) confer efficient XRN1 resistance and generate substantial amounts of sfRNAs. At the same time, SLIV-PK4 has lost its XRN1 resistance completely, while SLII-PK2 accumulated mutations that rendered it significantly less resistant to XRN1 digestion. Despite these mutations, PaRV retained its viability due to the production of sfRNAs from the other two copies of xrRNAs, xrRNA1 and xrRNA3. Based on these data, we concluded that xrRNA duplications improve viral fitness by providing a backup strategy for sfRNA production.Given the functional redundancy of sfRNA species produced from duplicated xrRNAs in ISFs, we propose two potential mechanisms of how this redundancy could benefit viral replication. The robust production of sfRNA is crucial for the efficient replication of flaviviruses. However, individual xrRNAs are dynamic structures that can transition between XRN1-resistant to XRN1 sensitive conformationDespite the clear benefit of more robust sfRNA production due to the structure duplications in the 3’UTR, there are two groups of flaviviruses (YFV serogroup and the majority of dISFs) that are evolutionary successful while lacking additional copies of canonical xrRNAs. Our results indicate that dISFs evolved a different strategy to backup sfRNA production. While all dISFs, except BinJV, don’t have additional copies of canonical xrRNA, we found that they all contain pseudoknot(s) formed by a conserved stem-loop elements (CS3/RCS3 in BinJv), which is not evident in other flavivirus clades. Our mutational analysis in the live BinJV demonstrated that the presence of the additional pseudoknots ensures the production of sfRNAs in amounts sufficient to prevent virus attenuation when the canonical pseudoknots of the corresponding xrRNAs are mutated. Curiously, novel pseudoknots of dISFS are not XRN1-resistant on their own and likely act by stabilising the resistant conformation of the upstream RNA elements. However, further structural studies are required to provide mechanistic insights on how these novel pseudoknots affect the folding of dISF xrRNAs. In particular, solving the crystal structure of dISF xrRNAs with and without nPKs will be desirable in the future to provide such information.15 and, through this study, are now also available for ISFs.The discovery of novel pseudoknots in dISFs and independent xrRNA duplication events in all flavivirus clades shows that flaviviruses evolved multiple strategies to ensure the reliable production of sfRNAs. Together with highly attenuated phenotypes of viruses completely deficient in sfRNA production, this further highlights the critical importance of sfRNAs for replication of all flaviviruses, including ISFs. Identifying the exact functions of sfRNAs in ISF-host interactions is the next step to understand their functional significance. This can be achieved by further exploring the biological effects of ISF sfRNAs in the loss of function model systems employing ISF mutants deficient in sfRNA production. We and others previously employed such systems to identify molecular functions of MBF sfRNAsAedes albopictus larvae cells C6/36 (ATCC – CRL-1660) and Aedes aegypti larvae cells Aag2 (ATCC – CCL-125) were obtained from the ATCC. Aedes albopictus larvae cells RML-1242 were a gift from Prof. Robert Tesh . C6/36 were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640). Aag2 cells were cultured in 1:1 mixture of Schneider’s Drosophila medium and Mitsuhashi & Maramorosch medium . RML-12 cells were cultured in Leibovitz’s L-15 medium supplemented with 10% tryptose-phosphate broth. All culture media were supplemented with 10% Fetal Calf Serum (FCS), 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM L-glutamine. Cells cultured at 28 °C with 5% CO2 or in sealed containers. All cell culture media and reagents were from Gibco, USA, unless otherwise specified.33. Mosquitoes were snap-frozen in liquid nitrogen and shipped for processing on dry ice. Total RNA was then isolated from individual mosquitoes and used for RT-PCR screening with Karumba virus (KRBV) genome primers KRBV-F and KRBV-R  by incubating cells with 50 μL of inoculum per cm2 of growth area for 1 h at 28 °C. Inoculated cells were then maintained in the growth medium containing a reduced amount of FCS (2%) to prevent overgrowth.Binjari (BinJV), Parramatta River (PaRV), Palm Creek (PCV) and Hidden Valley (HVV) viruses were previously isolated from Australian mosquito populations5 C6/36 cells grown in 96-well plates. After 2 h incubation with the inoculum, 180 µl overlay media was added to the cells and incubated at 27 °C in 5% CO2. The overlay media contained one part X M199 medium  and another part 2% carboxymethyl cellulose . At 3 days post-infection, cells were fixed with 100 µL/well of 80% acetone for 20 min at −20 °C, washed with PBS, thoroughly dried and blocked for 30 min with 150 µL/well of ClearMilk blocking solution . Cells were then incubated with 50 μL/well of mouse monoclonal antibody to flavivirus envelope protein , diluted in 1:100 for 1 h, followed by 1 h incubation with 50 μl/well of 1:800 dilution of goat anti-mouse IRDye 800CW secondary antibody . All antibodies were diluted with Clear Milk blocking buffer , and incubations were performed at 37 °C for 1 h. After each incubation with antibody, plates were washed 5 times with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBST). Plates were then scanned using an Odyssey CLx Imaging System (LI-COR) . Virus replication foci were counted using the Image Studio Lite software , and titres were determined based on dilution factors and expressed as focus forming units per mL (FFU mL^-1).Ten-fold serial dilutions of culture fluids were prepared in RPMI media supplemented with 2% FCS, and 25 µl of each dilution were used to infect 10260/OD280 and OD260/OD230 ratios.Total RNA was isolated from cells and mosquitoes using TRIreagent . Individual mosquitoes were homogenised in 500 μL TRIreagent for 5 min at 30 Hz using a Tissue Lyser II  before RNA isolation. Viral RNA from cell culture fluids was isolated using QIAamp Viral RNA Mini Kit . All RNA isolation procedures were conducted according to the manufacturer’s instructions. RNA concentrations were determined on NanoDrop One microspectrophotometer , and RNA purity was assessed by OD+ nylon membrane  for 90 min at 35 V in 0.5x TBE using the TransBlot Mini transfer apparatus  and UV-crosslinking at 1200 kDj/cm2. Membranes were pre-hybridised at 50 °C in ExpressHyb Hybridization Solution  for 1 h. The probes were prepared by end labelling 10 pmoles of DNA oligonucleotide complementary to the sfRNA  using T4 polynucleotide kinase  and purified from unincorporated nucleotides by gel filtration on Illustra MicroSpin G-25 Columns . Hybridisation was performed overnight at 50 °C in ExpressHyb Hybridisation Solution . After hybridisation, membranes were rinsed, washed 4 × 15 min with Northern Wash Buffer  at 50 °C and exposed to a phosphor screen  overnight. Signal detection was performed on Typhoon FLA 7000 Imager .Total RNA 2.5-10 μg) was mixed with an equal volume of Loading Buffer II , denatured at 85 °C for 5 min and chilled on ice for 2 min. Samples were then subjected to electrophoresis in 6% polyacrylamide TBE-Urea gels . Electrophoresis was performed for 90 min in 1x Tris-Borate-EDTA buffer pH8.0 (TBE). Gels were stained with ethidium bromide to visualise rRNA and documented using Omnidoc imager . RNA was then electroblotted onto Amersham Hybond-N.5-10 μg Ae. aegypti XRN1/pacman was generated by in vitro transcription (IVT). The DNA templates for IVT were prepared by amplifying a ~3 kb fragment of XRN1/pacman mRNA with primers designed to incorporate T7 promoter into PCR-products in either forward or reverse directions  following transfection in suspension protocol44. Cells were plated into 6-well plates and at 24 h post-transfection infected with PaRV, PCV or BinJV at MOI = 1. At 2 dpi cells were lysed in TRIreagent , and total RNA was isolated.The dsRNA against Total RNA was isolated from C6/36 cells infected with PaRV, PCV, BinJV or HVV at 5dpi and from KRBV-positive mosquitoes, and RT-PCR was used to amplify the fragment of the viral genome containing 3’UTR and the small terminal part of the NS5 gene. RT-PCR was performed using SSIII One-Step RT-PCR Kit with Platinum Taq  according to the manufacturer’s recommendations. PCR primers . 3’UTRs were in vitro transcribed from 1 μg of linearised plasmids using MEGAscript T7 Transcription Kit  according to the manufacturer’s recommendations. RNA was purified by LiCl precipitation and analysed by electrophoresis in a 1.2% denaturing agarose gel. RNA was then refolded in NEB3 buffer by heating at 85 °C for 5 min followed by gradual cooling to 28 °C. The refolded RNA (1 μg) was incubated with 1U XRN1  and 10U RppH  in 20 μL of reaction mixture containing 1x NEB3 buffer  and 1 u/μL RNasin RNase Inhibitor . Incubation was performed for 2 h at 28 °C. The reaction was stopped by adding 20 μL of Loading Buffer II , heating for 5 min at 85 °C and placing on ice. The entire volume was then loaded into 6% polyacrylamide TBE-Urea gels , and electrophoresis was performed for 90 min in 1xTBE. Gels were stained with ethidium bromide and documented using an Omnidoc imager .Total RNA from infected C6/36 cells 20 μg) was denatured by heating at 75 °C for 5 min, then placed on ice. Denatured RNA was circularised by incubation with 10U T4 RNA Ligase 1  in 20 μL of the reaction mixture containing 1x T4 RNA Ligase Buffer , 1 mM ATP , 12.5% PEG8000 and 1 u/μL RNasin RNase Inhibitor . Incubation was performed O/N at 16 °C, then 10 μL of the mix was used as the template for RT reaction with LigSeq-RT primer ). The solution was equally divided between two tubes (72 μL each) and incubated for 30 min at 28 °C with either 8 μL of 50 mM NMIA  in DMSO ( + reaction) or 8 μL of DMSO (- reaction). RNA solutions were mixed with 4 μL 3 M NaOAc pH 5.2, 2 μL 100 mM EDTA, 1 μL Glycogen (20 mg/mL) and 350 μL ice-cold 100% ethanol and incubated overnight at −80 °C to precipitate RNA. RNA was pelleted by 30 min of centrifugation at 10,000 x g, 4 °C, washed with 75% DEPC-EtOH and resuspended in 10 μL 0.5x TE buffer pH 8.0. Five microliters of each RNA solution were combined with 6 μL DEPC-H2O and 1 μL of 5’-FAM-labelled SHAPE primer  and 4 μL SSIII First Strand Buffer . RT reaction was performed at 52 °C for 30 min and quenched by adding 1 μL 4 M NaOH followed by incubation at 95 °C for 3 min. NaOH was neutralised by adding 2 μL of 2 M HCl. Reactions were subsequently chilled on ice and EtOH-precipitated overnight at −80 °C as described above. Pelleted cDNA was dissolved in 40 μL of deionised formamide at 65 °C for 10 min and then stored at −80 °C.In vitro transcribed 3’UTR RNA 20 μg) dissolved in 20 μL DEPC-H0 μg diss2O, 1 μL 10 mM dNTPs, 1 μL of 10 mM ddTTP , 1 μL SuperScript III RT  and 4 μL SSIII First Strand Buffer . The cDNA was then synthesised and precipitated as described above for the SHAPE reactions. Pelleted cDNA was dissolved in 40 μL of deionised formamide at 65 °C for 10 min, and 10 μL of the solution was combined with 40 μL of (+) and (-) SHAPE cDNAs. Samples were then analysed by high throughput capillary electrophoresis. Chromatogram data was analysed, and SHAPE reactivities were computed using QuShape software45. Mean SHAPE reactivities from three independent experiments were used as constraints for guided RNA folding with RNAStructure software (https://rna.urmc.rochester.edu/RNAstructureHelp.html). The folding temperature was set to 28 C, folding of pseudoknots was enabled, and the maximum distance of base pairing was set to 100 nt. Resultant secondary structures were visualised using VARNA v3.93. Noncanonical C-A pairing in cISF sfRNAs was forced manually.To prepare a sequencing ladder, 10 μg RNA was incubated with 10 pmol 5’-HEX-labelled SHAPE primer (Supplementary Table 46 web server (http://rtips.dna.bio.keio.ac.jp/ipknot/). Secondary structures of AnFV 3’UTR elements were predicted using mfold v2.3 (http://www.unafold.org/mfold/applications/rna-folding-form-v2.php), and pseudoknots were located manually. The folding temperature was set to 28 °C, and the maximum distance between paired nucleotides was limited to 150. All secondary structures were visualised using VARNA v3.93.Pseudoknots if dISF xrRNAs were predicted using IPknot47. Experimentally determined or predicted secondary structures were provided for each xrRNAs as structural constrains (#S option). Stockholm formatted alignments and consensus secondary structures generated by LocARNA were used to build covariance models followed by evaluation and visualisation of consensus structures with R-scape software36 (http://eddylab.org/R-scape/). Consensus pseudoknots were specified manually in R-scape input as consensus structures #CS_2 and #CS_3.Structure-based multiple alignments of xrRNAs were performed using RIBOSUM-like similarity scoring implemented in the LocARNA package (Freiburg RNA Tools)48 for both the polyprotein (G-INS-i) and complete 3’UTR  and (1655 × 48) for the polyprotein and nucleotide, respectively, and accession IDs are available from Supplementary Table 50 were used to construct the consensus maximum-likelihood phylogenetic inference . The best-fit protein substitution model (LG + F + R6) and nucleotide substitution model (GTR + F + I + G4) was selected using the ModelFinder program within IQ-TREE2 informed using the Bayesian Information Criterion. The resultant consensus tree was visualised using FigTree v1.4.4 .Multiple sequence alignment (MSA) used for phylogenetic inference were constructed using iterative refinement methods incorporated into MAFFT v7.475 48) and 655 × 48 Plasmid constructs based on pUC19 vector containing WT 3’UTR of each ISF were used as templates for PCR-directed site-specific mutagenesis, which was performed using Q5 Site-Directed Mutagenesis Kit  with mutagenesis primers listed in Supplementary Table Mutant viruses were assembled using circular polymerase extension reaction (CPER) according to the established protocols for PaRV, PCV and BinJV. CPER fragments containing WT or mutated or 3’UTRs were amplified from the plasmids using PrimeStar GXL polymerase  and gel-purified. Infectious cDNA from CPER assembly was transfected into C6/36 cells using Lipofectamine 2000  according to the manufacturer’s instructions. Culture fluids of transfected cells were then sampled for virus titration at 5, 7 and 10 days post-transfection. To confirm the presence of the mutations in 3’UTRs, viral RNA was isolated from culture fluid samples using QIAamp Viral RNA Kit  and used as a template for RT-PCR with 3’UTR sequencing primers  were then harvested at 3, 5 and 7 days post-infection and subjected to foci-forming immunoassay to determine the virus titres, from which growth curves were plotted.The growth kinetics of wild type and mutant viruses were assessed in RML-12 cells. Cells were seeded at 2 × 10Statistical analyses were performed using GraphPad Prism v.9.0. Exact statistical tests are specified in figure legends.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


KRAS, SYNE1, CACNA1H, PCLO, FBXL2, and DNAH11. The main CNV events underwent clonal-clonal evolution (20q amplification (amp), 17p deletion (del), 18q del and 8p del), subclonal-clonal evolution  and metastasis-specific evolution (8q amp) during the process of CLM. In addition, we revealed a potential mechanism of tumor cell immune escape by analyzing human leukocytes antigens (HLA) related clonal neoantigens and immune cell components in CLM. Our study proposed a novel liver metastasis-related evolutionary process in colorectal cancer and emphasized the theory of neo-immune escape in colorectal liver metastasis.Liver is the most common site where metastatic lesions of colorectal cancer (CRC) arise. Although researches have shown mutations in driver genes, copy number variations (CNV) and alterations in relevant signaling pathways promoted the tumor evolution and immune escape during colorectal liver metastasis (CLM), the underlying mechanism remains largely elusive. Tumor and matched metastatic tissues were collected from 16 patients diagnosed with colorectal cancer and subjected to whole-exome sequencing (WES) and RNA sequencing (RNA-seq) for studying colorectal cancer clonal evolution and immune escape during CLM. Shared somatic mutations between primary and metastatic tissues with a commonly observed subclonal-clonal (S-C) changing pattern indicated a common clonal origin between two lesions. The recurrent mutations with S-C changing pattern included those in  The colorectal liver metastasis (CLM) is a multistep and complex process during which tumor cells develop aggressive phenotypic features, and intensely interact with the host immune microenvironment . ApproxiRAS, TP53, and SMAD4 were related with worse prognosis [3) [APC, KRAS, NRAS, TP53, or BRAF were shared in primary and synchronous liver metastases while SMAD4 and PIK3CA were private driver mutations in metastases [APC splice-site alterations in intronic regions and large in-frame deletions in CTNNB1 [Recent researches have highlighted the importance of cancer-specific genetic alterations, mutations in pathways, microsatellite status in predicting CLM, and postoperative survival –6. Yoshiosis [3) . Anothern CTNNB1 . HoweverTraditionally, cancer was proposed to be a disease associated with dynamic gene alterations, while increasing studies demonstrated that the immune system also plays a critical role in tumor progression . Immune To obtain comprehensive understanding of the clonal evolution and tumor cell immune escape in course of CLM progress, we performed WES (30 samples) and RNA-seq (27 samples) from 16 patients with CRC and matched liver metastases, and identified new metastasis-associated evolutionary patterns and illuminated tumor immune-related genetic changes in CLM.APC, AXIN1, AMER1, TCF7L2, and KMT2D, mutated in 80% patients (12/15) and genes in HGF/MET signaling pathway including KRAS, COL5A1 and HGF, mutated in 60% patients (9/15)  and 9996 small insertions and deletions (Indels) in those samples. In result, genes with high mutational frequency were components of WNT-β-catenin signaling pathway such as 15) Fig. . Moreove15) Fig. . We furt15) Fig. , C, indi15) Fig. . Major CAPC was the highest in both primary tumor and metastases, indicating that APC mutation was involved in liver metastasis in addition to tumorigenesis [TP53 was higher than KRAS in primary tumor, agreeing with the previous conclusion that mutations in TP53 occur earlier than KRAS mutations in primary tumors [KRAS mutations occur earlier than TP53 in liver metastasis. Moreover, in liver metastases, the top-10 mutations with the highest median CCFs were associated with calcium channel or cell-adhesion functions such as CACNA1H, PCDHA2, PCDHB15, RYR1, FAT4, indicating tumor cell mobility mediated by Ca2+ [To explore the probable timing order of the mutation events arose in CLM, we analyzed the distribution of the cancer cell fractions (CCF) in samples , where higenesis . In addiy tumors , while K by Ca2+  might plP = 0.03)  and patients (n = 19) with CRC distant metastasis and OS of <5 years (M1& <5 Y) from . As shown in Fig. KRAS, SYNE1, CACNA1H, PCLO, FBXL2, DNAH11 , in only metastatic samples of three patients . Analysis of TCGA (Firehose Legacy) samples showed SYNE1 mutation was closely associated with metastatic patients’s OS (n = 153) (P = 0.002) but was not significantly correlated with survival of non-metastatic patients (n = 465) (P = 0.0714)    Fig. . The mRN11) Fig. . HoweverGA) Fig. .Fig. 2PhTP53 (17p del), SMAD2, SMAD4(18q del) and MYC, CNBD1, HEY1, RUNX1T1, CDH17(8q amp)    between primary tumors and liver metastases. The evolutionary patterns from primary to liver metastases were shown in Fig. mp) Fig. . High mRcy) Fig. . In addicy) Fig. . The frecy) Fig. . These rP = 0.009) than clonal in primary lesions, while in liver metastases, subclonal and clonal mutation loads had no significant difference (P = 0.557). One possible reason is the bottleneck effects in the process of tumor evolution and selection during metastasis. The proportion of clones mutation was increased compared to subclonal mutation in metastatic lesions due to metastasis specifically selected certain tumor clones. Subclonal mutation loads in primary tumors were higher than metastasis while the clonal mutation loads were lower , while LOHHLA in liver metastases has increased tendency. Although both TMB and HLA-corrected TMB had no significant difference between primary and liver metastasis, HLA-corrected TMB increased the P value from 0.41 to 0.26  is an indicator to evaluate the efficacy of immunotherapy , but a nP = 0.62), while the count of clonal neoantigens was higher than subclonal in liver metastasis (P = 0.0025). Moreover, the prevalence of neoantigen-located LOHHLA of liver metastases was significantly higher than in primary tumors (P < 0.001). Also, in liver metastases, numbers of clonal and subclonal neoantigens increased compared with primary tumors . The numbers of non-expressed neoantigens in both clonal and subclonal groups were significantly higher than that of expressed neoantigens, especially in the subclonal group (P = 0.007). Taken together, under the pressure of immune selection and seeding, the HLALOH and the deletion of expression of neoantigens occurred during the process of tumor evolution and metastasis.The distribution of clonal/subclonal neoantigens in all samples were shown in Fig. ors Fig. . The mRNP < 0.001), indicating human immunity defects in liver metastases; primary tumor had the most cancer associated fibroblasts compared to normal and metastatic samples ; CD4+ T cells, CD8+ T cells and endothelial cells were significantly decreased in liver metastasis than in normal tissues or primary tumors (p < 0.001), while macrophages increased significantly in liver metastasis (p < 0.001)  Fig. . These rTumor malignant progression is a dynamic evolutionary process, researchers sought to explore the genetic features of tumors across various stages , distingAPC mutation was only observed in the metastatic samples, and CCF analysis showed APC mutation was an early event in liver metastasis, indicating that APC would have a function of facilitating liver metastasis. The average percentage of liver metastasis-private mutation is higher than of primary-private mutations, this tendency was consistent to what has been previously described [2+ channel functions, including mutations in CACNA1H, PCDHA2, PCDHB15, RYR1, and FAT4, indicating the potential components of cell mobility in early liver metastasis.In our study, CRC primary and liver metastasis had the common ancestor at the mutational level, meanwhile had their private features Fig. . We founescribed , howeverescribed , 27, notSYNE1, a mutated gene with S-C clonal evolution, had a high prevalence of mutation in fuctional region which was observed only in liver metastases in 20% patients with CRC and lower expression in liver metastatic lesions. However, the SYNE1 mRNA level in primary CRC was not correlated with unfavorable prognosis, indicating functional mechanism of SYNE1 in CLM was obscure, whether there was synergistic effect between gene mutation or protein level of SYNE1 in CLM process should be further investigated in future studies. Other studies have also confirmed that abnormal methylation of SYNE1 promoter is closely related to CRC [One highlight in our study is the finding that mutated genes with S-C clonal evolution from CRC primary tumor to liver metastases are enriched in cell mobility-related ECM signaling pathways and were associated with metastasis by cohort analysis Figs. ,3. Subcld to CRC , and is d to CRC .SMAD2 and SMAD4 were located on Chr 18q. Another early event is Chr 17q del and TP53 is a key driver gene on this segment, TP53 is a prevalently mutated gene and closely associated with CRC malignancy [MYC, CNBD1, HEY1, RUNX1T1, CDH17, high expression of HEY1 gene promotes self-renewal of tumor stem cells, especially in liver cancer [PXDNL with significantly different mutational frequency was associated with the CRC prognosis. These finding enriched the knowledge for understanding CLM progression.In CNV events level, we firstly confirmed the different clonal patterns of major CNV events. Moreover, we explored the probable timing order of the CNV events from CRC primary to matched liver metastases according to CNV clonality. Clonal pattern of C-C were early events, 0-C defined as liver metastasis-private were later events, and S-C were defined as middle-stage events Fig. . Among elignancy . The corr cancer . On liveKRAS, SYNE1, FBXL2, DNAH11 and CACNA1H, indicating this mutational clonal patter is facilitating tumor cell to escape immune monitor. Besides, the different CRC stages have distinct immune microenvironment, the CD4+, CD8+ T cells were significantly decreased and TAMs significantly increased in liver metastases, indicating T cell mediating immune responses was inhibited when TAM induced tumor cell metastasis by promoting angiogenesis [At present, it is difficult to completely understand the process of immune escape of tumor cells in CLM. Immune microenvironments were varied both between and within tumors as well as in various stages during tumor evolution. We found the incidence of HLALOH and the number of clonal neoantigens significantly increased in liver metastasis. The HLALOH and deletion of expressed neoantigens may be a potential mechanism of CRC cell immune escape in agree with Rosenthal R et al.’s report in lung cancer . Howeverogenesis , and proogenesis . In addiogenesis . Taken tIn summary, our data exhibited a new metastasis-associated clonal evolutionary pattern at somatic SNV and CNV levels, revealed immune microenvironment in CLM and explored the potential mechanism of immune escape via LOH of HLA. Our results provide a new experimental basis for understanding CLM.Sixteen patients with CRC were enrolled from Daping Hospital, Army Medical University  from 2015 to 2019. Patients were either untreated or treated with neoadjuvant therapy containing oxaliplatin, Irinotecan or bevacizumab. The detailed clinicopathological and sample information were shown in Supplementary Table SYNE1 mutated CRC patients were obtained from public databases of The Cancer Genome Atlas   and analyzed.Online analysis of TCGA datasets including clinical and SNVs data of 77 patients (1). without CRC metastasis and (2). with OS of more than 5 years (M0& >5 Y), 19 patients with (1). CRC distant metastasis and (2). OS of <5 years (M1& <5 Y), and Clinical and SNVs data of Primary tumor with matched normal and liver metastasis tissues were collected and prepared into formalin-fixed paraffin-embedded (FFPE) samples. Genomic DNA extraction was performed with the TIANamp Genomic DNA kit  following manufacturer’s instruction. The purity and concentration of DNA were determined using Nanodrop 2000 spectrophotometer and Qubit 2.0 Fluorometer with Quanti-IT dsDNA HS Assay Kit . Library construction was then performed using a custom 53 M length capturing probe, made by Integrated DNA Technologies , and covering the coding regions of all genes and partial non-coding regions. Captured libraries were then pair-end sequenced in 100 bp lengths with Geneplus-2000 sequencing platform  following the manufacturer’s guidance. Raw data from next-generation sequencing was then filtered to remove low-quality reads and adaptor sequence. Reads were further mapped to the reference human genome (hg19) utilizing BWA aligner (version 0.7.10) for mutation calling. In total, 15 pairs of primary and metastasis samples were subjected to WES with a mean sequencing depth of 150×. Matched DNA from normal colorectal tissue was used as control.The software of ABSOLUTE (version 1.2) was used to evaluate the tumor purity and genomic ploidy by analyzing CCF and allele-specific copy-numbers of each sample . To ensuhttps://evolution.genetics.washington.edu/phylip/phylipweb.html) with the maximum parsimony method to infer the phylogeny of multiple specimens from individual patients based on the presence or absence of SNVs and indels. FigTree (version 1.4.3)  was used to visualize the reconstructed trees.We used PHYLIP (version 3.6)  [The HLA LOH events in tumor were identified with the method by McGranahan N. et al., HLA-LOH was defined when one of the two alleles of HLA gene was below 0.5, and there was significant difference between the log copy ratio of the two alleles ( < 0.01) .Primary tumor with matched normal and liver metastasis tissues were collected and prepared into FFPE samples. RNA extraction, sequencing library construction, sequencing and FASTQ data quality control were performed in accordance with the protocol by Nick D.L. Owens et al. , alterathttps://icbi-lab.github.io/immunedeconv/ website [Based on RNA-seq data, we calculated the immune cell scores in normal, primary and matched liver metastasis region to explore the different immune microenvironment status and immune infiltration in these tissues with the method stated in  website . The immP < 0.05 for paired two-tail student t tests.Chi-Square test or the Fisher’s exact test was employed to calculate the significant difference in categorical variables between two groups, while Wilcoxon Mann–Whitney test was performed for comparing continuous variables. R software (version 3.6.1), Linux sever equipped with Python (version 2.7.13) or GraphPad Prism software (version 8.0.2) were employed to analyze data and carry out visualization. Statistical significance was defined as supplementary table 1Figure S1Figure S2Figure S3Supplementary figure legends


Therefore, an RNA editing deficiency at the Q/R site in GluA2 due to the downregulation of ADAR2 in the motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients suggests that Ca2+-permeable AMPA receptors and the dysregulation of RNA editing are suitable therapeutic targets for ALS. Gene therapy has recently emerged as a new therapeutic opportunity for many heretofore incurable diseases, and RNA editing dysregulation can be a target for gene therapy; therefore, we reviewed neurological diseases associated with dysregulated RNA editing and a new therapeutic approach targeting dysregulated RNA editing, especially one that is effective in ALS.The conversion of adenosine to inosine in RNA editing (A-to-I RNA editing) is recognized as a critical post-transcriptional modification of RNA by adenosine deaminases acting on RNAs (ADARs). A-to-I RNA editing occurs predominantly in mammalian and human central nervous systems and can alter the function of translated proteins, including neurotransmitter receptors and ion channels; therefore, the role of dysregulated RNA editing in the pathogenesis of neurological diseases has been speculated. Specifically, the failure of A-to-I RNA editing at the glutamine/arginine (Q/R) site of the GluA2 subunit causes excessive permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors to Ca Morhreshold ,144. Howhreshold ,146. TheA-to-I RNA editing-based therapeutic strategies would effectively rescue the death of lower motor neurons in the majority of ALS patients, including patients with both sporadic and some with familial ALS that are associated with the dysregulation of A-to-I RNA editing. However, as the roles of dysregulation of RNA editing in the upper motor neuron death remain elusive, further studies are needed for the elucidation of the pathogenic mechanism underlying the death of the upper motor neurons in ALS and, more broadly, the death of the cortical neurons in frontotemporal lobar degeneration, which is closely associated with ALS. When such a new therapeutic strategy is realized in ALS, disease-specific biomarkers will also be increasingly required. Since biomarkers linked to disease-specific molecular changes can specify ALS, the demonstration of deficient ADAR2 activity non-invasively will help immensely in the evaluation of treatment efficacy. As the RNA editing efficiency at the ADAR2 sites in extracellular RNAs reflects intracellular ADAR2 activity in vitro , demonst2+ influx through Ca2+-permeable AMPA receptors seems to be a plausible pathomechanism in ALS, the development of therapy based on this underlying pathomechanism would change the currently incurable ALS to a treatable disease. Moreover, we will be able to evaluate therapeutic response noninvasively by measuring alterations in editing efficiency in the RNAs in body fluids.We reviewed the mechanistic roles of defective RNA editing in neurological diseases and the potential therapeutic strategies targeting RNA editing in these diseases, especially for ALS. As excitotoxicity mediated by excessive Ca


Hepatitis A virus (HAV) is a causative agent of acute hepatitis and can occasionally induce acute liver failure. However, specific potent anti-HAV drug is not available on the market currently. Thus, we investigated several novel therapeutic drugs through a drug repositioning approach, targeting ribonucleic acid (RNA)-dependent RNA polymerase and RNA-dependent deoxyribonucleic acid polymerase. In the present study, we examined the anti-HAV activity of 18 drugs by measuring the HAV subgenomic replicon and HAV HA11-1299 genotype IIIA replication in human hepatoma cell lines, using a reporter assay and real-time reverse transcription polymerase chain reaction, respectively. Mutagenesis of the HAV 5’ untranslated region was also examined by next-generation sequencing. These specific parameters were explored because lethal mutagenesis has emerged as a novel potential therapeutic approach to treat RNA virus infections. Favipiravir inhibited HAV replication in both Huh7 and PLC/PRF/5 cells, although ribavirin inhibited HAV replication in only Huh7 cells. Next-generation sequencing demonstrated that favipiravir could introduce nucleotide mutations into the HAV genome more than ribavirin. In conclusion, favipiravir could introduce nucleotide mutations into the HAV genome and work as an antiviral against HAV infection. Provided that further in vivo experiments confirm its efficacy, favipiravir would be useful for the treatment of severe HAV infection. The hepatitis A virus (HAV) genome consists of a single-stranded, positive-sense RNA, approximately 7.5 kilobases in length, encoding a large open reading frame (ORF) that is flanked by highly conserved 5′ and 3′ untranslated regions (UTRs), and encodes four structural proteins  and seven nonstructural proteins . Initiation translation of HAV RNA is mediated through the HAV internal ribosome entry site (IRES) element located mainly in the 5’ UTR. It is reported that the HAV 3D protein is a viral ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), and HAV replicates through genomic plus-stranded RNA . Variables indicate a higher concentration of two values that sandwich IC50 (A), a lower concentration of two values that sandwich IC50 (B), HAV RNA levels (%) at B (C), and HAV RNA levels (%) at A (D)  .t test. p < 0.05 was considered significant. All assays were performed in triplicate.Data are expressed as the means ± standard deviations (SD). Statistical analyses were performed with Student’s We demonstrated that favipiravir effectively suppressed HAV replication through the introduction of mutagenesis into the HAV genome and that favipiravir could be useful for the control of HAV infection.


An overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications. Advanced sequencing technology that expeditiously provides whole-genome sequences of pathogens has allowed PCR-based platforms to be a nimble tool in diagnosing emerging novel infections. The most recent and high-profile use of PCR assays is the molecular identification of SARS-CoV-2 for rapid laboratory diagnosis of Covid-19, a pandemic that has overwhelmed public healthcare globally.Rapid and accurate diagnosis of infectious diseases is invaluable for early therapeutic intervention and effective treatment and is critical for infection control that can have serious repercussions to public health outcomes. Serological and molecular assays are fast superseding cell culture-based methods as timely diagnostic tools in infection surveillance. Due to its high sensitivity of detection, specificity for causative agents, broad dynamic range, and its speed and ease of use, quantitative polymerase chain reaction (qPCR) has become the gold standard procedure in clinical diagnosis, particularly in the diagnosis of viral diseases, such as the arboviruses, hepatitis viruses and HIV2. While undoubtedly streamlined for user convenience, interruption in the supply of any of the components in the integrated system due to limited manufacturing capacity or surge demand can greatly hamper diagnostic capabilities as epitomised by the acute global shortage of kits during the Covid-19 pandemic3. Apart from supply chain strain, kit-based approaches also present another challenge—the affordability of the kits and their dedicated instruments, which can be prohibitive to low and low-middle income countries in South Asia, South America and Africa.Typically, a clinical diagnostic approach involves viral RNA extraction from human biological samples followed by PCR assay targeting virus-specific nucleic acid. Viral RNA is routinely extracted using commercially available solid-phase RNA extraction kits that are often supplied by a handful of major manufacturers who vertically integrate these kits with their proprietary instruments and essential plastic wares, leaving little or no room for generic suppliers4. Nonetheless, even where automated liquid handling capabilities are lacking, the adapted protocol for a 96-well format can still be carried out manually using a multi-channel pipette, albeit at a slower pace yet remaining cost-effective for acquisition of RNA from large numbers of samples.To ensure that mass testing in future epidemics is not compromised by the unavailability and/or unaffordability of RNA extraction kits, a robust alternative for RNA extraction that guarantees a stable and scalable supply at low cost while maintaining diagnostic sensitivity and specificity, is acutely needed. Extremely versatile and robust, TRIzol-based reagents have widely been used for isolating high quality RNA from a variety of biological samples including cells, tissues, serum, blood and microbes. The powerful protein denaturant effectively stabilizes RNA and inactivates RNases and infectious agents, allowing the recovery of RNA from very small amount of input samples. However, RNA isolation from a high volume of samples in the conventional 1.5-mL microcentrifuge tube format is incompatible with high-throughput applications. Here we demonstrate that a well-established, TRIzol-based, non-kit RNA extraction protocol can be adapted into a semi-automated, high-throughput format using equipment readily available in research and hospital laboratories. Moreover, recent establishment of Biofoundries around the world, with their versatile and scalable automation pipelines, is poised to lend firm infrastructural support to our approach7. As such, saliva and pharyngeal swabs were used as diagnostic samples in this study due to the ease of self-collection from volunteers, negating the need for skilled personnel for sample collection. Importantly, saliva can be highly viscous and difficult to pipet and was presented as an assessment on the automated liquid handlers’ capability to exact extraction from specimens with varying viscosity. For comparison, analyses were also carried out on oropharyngeal nasal mid-turbinate (OP-NMT) swabs performed by a trained medical personnel. Two laboratory automation platforms were employed, namely, the Opentrons OT-2 lab robot and the Eppendorf epMotion® 5075, both which were equipped with an 8-channel volumetric dispensing arm and a thermal block for 96-well plates.The TRIzol method, while widely proven effective in RNA extraction, poses challenges to adaptation for high-throughput processing performed on automated liquid handling instruments due to the multiple steps involved. The need for phase separation, precipitation by centrifugation and potential phenol carryover further complicates the workflow. In this study, the conventional protocol was adapted to a 96-well format with emphasis on miniaturization and reduced human intervention Fig. . To valiIn order to perform RNA isolation in 96-well format, the reaction volume was scaled down proportionally in a 3:1 TRIzol reagent-to-sample ratio, reducing the volume of reagent required by sevenfold to 105-μL, thereby directly lowering the consumable cost per sample extraction. With a concomitant decrease in input sample to a volume of just 45-μL, glycogen was added as a co-precipitant to aid in RNA recovery. In a kit-free extraction method, precipitation and purification of RNA by centrifugation remain ineludible steps, making manual intervention obligatory in the workflow. The addition of glycogen as an inert carrier for nucleic acids was crucial in overcoming the challenge of low centrifugal force when using swing-out rotors for plates. The adapted procedure successfully extracted 1 to 4 ng/μl of RNA from both saliva and throat swabs with the yield slightly higher from saliva Fig. A.Figure During transfer of the aqueous phase following phase separation, cross-contamination with proteins, lipids, DNA and phenol can occur. Indeed, ensuring high quality of isolated RNA, particularly in a miniaturized format, proved challenging. Phenol carryover was identified in sample extractions using both automated liquid handlers as indicated by a characteristic absorbance maximum at 270 nm. Despite the relatively low purity and quantity, the extracted RNA was tested for its suitability for use in sensitive downstream applications such as real time RT-qPCR. The phenol contamination did not appear to interfere with the efficacy of the RT-qPCR targeted at the ubiquitous human Ribonuclease P (RNase P) from both the saliva and throat  and nCoV-N1 (Ct = 30.6).To assess viral RNA extraction efficiency of the miniaturised, semi-automated TRIzol-based extraction protocol against commercial RNA purification kits, biological samples were spiked with heat-inactivated 2019 novel coronavirus (nCoV) and RNA extracted using the QIAamp Viral RNA Mini Kit specific for viral RNA isolation from body fluids, the RNeasy Mini Kit for generic purification of total RNA from cells as well as the TRIzol-based method performed manually at standard volume. To ensure consistent sample input, 45 ul of biological samples were each spiked with a fixed quantity of 4,500 copies of nCoV, and topped up with DMEM to the varying starting sample volumes recommended for each kit. Kit-extracted RNA was eluted according to manufacturer’s instructions, followed by volume reduction by evaporation to 10 ul to standardize with that used in the miniaturised TRIzol-based method before performing RT-qPCR assay targeted at RNaseP and SARS-CoV-2 N gene. The CDC 2019-nCoV N1 primer set was used as it has been shown to be more sensitive than the N2 or N3 sets, typically generating lower Ct values from positive samplesely Fig. . The res1–105 copies per reaction) of SARS-CoV-2 VLPs and nCoV. The highest quantified concentration in the standard curve for VLPs was limited to 104 copies per reaction as the available stock concentration was 1700 copies per μL. The extracted RNA showed a detection limit of 10 copies of VLP per reaction, while that of nCoV was 100 copies per reaction, suggesting good efficiency of extraction using the high-throughput platform  was detected out of the expected 16 positive spiked samples  in the negative, water-spiked controls, underlining the shortcoming in specificity of SYBR Green-based detection, particularly for diagnostic analyses . All experiments were performed in accordance with NUS OSHE guidelines and regulations. Informed consent was obtained from all volunteers.10. After collection, the swab was immediately suspended in 400 μl Dulbecco’s Modified Eagle Medium  and mixed vigorously to transfer the specimen. Saliva collection was carried out by briefly swirling the saliva in the mouth, pooling and spitting into a sterile 5 ml tube. Oropharyngeal nasal mid-turbinate swabs were conducted by a medically trained healthcare worker using a sterile polyester swab with a polystyrene shaft  which was suspended directly in 400 μl Dulbecco’s Modified Eagle Medium  and mixed vigorously to transfer the specimen. Consumption of food, water and brushing of teeth was avoided at least an hour before sampling. All samples were kept on ice until RNA extraction on the same day.The pharyngeal, or throat, swab was performed using a sterile polyester swab with a polystyrene shaft  according to instructions detailed in Won et al. 20208 and heat-inactivated 2019 novel coronavirus  with serial dilutions made using diethylpyrocarbonate (DEPC)-treated water as diluent. For mock SARS-CoV-2 positive samples in the 24 healthy volunteers, both saliva and throat samples from 8 volunteers were spiked with VLPs and another 8 volunteers with nCoV at 1 × 105 copies/ml concentration before RNA extraction. Separately, the oropharyngeal nasal mid-turbinate samples were spiked with 1 × 104 and 1 × 105 copies/ml nCoV for 8 volunteers each.Positive controls for SARS-CoV-2 were prepared using MS2 bacteriophage SARS-CoV-2 virus-like-particles  provided by Crone et al. 2020g for 15 min at 4 °C, 80 μl of the clear, upper aqueous layer was aspirated, transferred to a clean well containing 3 μl glycogen (2 μg/μl) and mixed slowly by pipetting 5 times. An equal volume, or 80 μl, of isopropanol was added next, mixed slowly by pipetting 20 times and allowed to incubate for 10 min at room temperature before centrifugation at 3200 × g at 4 °C for 20 min. The supernatant was aspirated and the remaining pellet washed with 150 μl of 75% ethanol by slowly pipetting 5 times before centrifugation again at 3200 × g at 4 °C for 10 min. The washing step was carried out twice and the pellet dried at 65 °C for 5 min on a heating block. To solubilise the RNA pellet, 10 μl of DEPC-treated water pre-warmed to 65 °C was added and mixed slowly by pipetting 20 times. The RNA suspension was allowed to incubate for 10 min at room temperature to further improve solubility before use in PCR analysis. The RNA concentration was determined with a microplate reader  using the Quant-iT RNA assay kit .Total RNA was extracted from throat, saliva and oropharyngeal nasal mid-turbinate samples using TRIzol  with modifications optimised for a 96-well format ready for multi-channel pipette or automated liquid handlers. Automated pipetting was performed using an 8-channel pipetting arm of the OT-2 lab robot  and epMotion 5075 . For each extraction, 45 μl of sample was aliquoted into a 96-well PCR plate and 105 μl of TRIzol was added and mixed vigorously by pipetting up-and-down 20 times. After incubation for 10 min at room temperature, 60 μl chloroform was added and mixed vigorously by pipetting 20 times before incubating for another 3 min. After centrifugation on a plate rotor  at a maximum speed of 3200 × RNA purification by commercial kits, Qiagen QIAamp Viral RNA kit and RNeasy Mini kit, was carried out according to manufacturer’s instructions using the recommended starting sample volume and final elution volume. The volume of the RNA elute was brought down to a standardised volume of 10 ul by vacuum centrifugation before RT-qPCR analysis.For SYBR Green-based detection, RT-qPCR was carried out using Luna Universal One-Step RT-qPCR Kit , iTaq Universal SYBR Green One-Step Kit  and KAPA SYBR FAST One-Step qRT-PCR Master Mix Kit . In the reaction setup for all kits, 1 μl of extracted RNA was added to half the standard reaction, i.e. 10 μl, volume. The following primer sets were used: human RNase P , US CDC SARS-CoV-2 N1 . RT-qPCR was performed following manufacturer’s thermal cycling protocol using the Bio Rad CFX96 Real-Time PCR Detection System  with the threshold setting determined by the CFX Maestro software. In addition, qualitative detection specific for SARS-CoV-2 was conducted using the MiRXES Fortitude Kit 2.0 RUO , which is a one-step, multiplex real-time RT-PCR kit with three primer–probe sets targeting the ORF1ab region 1 and 2 of the SARS-CoV-2 genome as well as an internal control. The PCR reaction setup and run profile was carried out according to manufacturer’s instruction with the recommended threshold setting at 50.All data generated or analysed during this study are included in this published article (and its Supplementary Information file).Supplementary Information.