Many habitat-building corals undergo mass synchronous spawning events. Yet, despite the enormous amounts of larvae produced, larval dispersal from a single spawning event and the reliability of larval supply are highly dependent on vagaries of ocean currents. However, colonies from the same population will occasionally spawn over successive months. These split spawning events likely help to realign reproduction events to favourable environmental conditions. Here, we show that split spawning may benefit corals by increasing the reliability of larval supply. By modelling the dispersal of coral larvae across Australia’s Great Barrier Reef, we find that split spawning increased the diversity of sources and reliability of larval supply the reefs could receive, especially in regions with low and intrinsically variable connectivity. Such increased larval supply might help counteract the expected declines in reproductive success associated with split spawning events. Corals occasionally split their spawning over two consecutive months rather than utilising a single annual event. Here, the authors model coral larval dispersal to show that split spawning may increase the reliability of larval supply to reefs, with implications for recovery from disturbances. In marine environments, biogenic habitat providers — including seagrasses, kelps, and corals — host thousands of species that benefit economies through ecosystem function5. For coral reefs, in particular, recent climate-related disturbances have vastly reduced coral populations through mass coral bleaching6, compounded by local pressures including storms and pest outbreaks7. Once damaged, a key driver to coral recolonization and subsequent habitat recovery is the supply of larvae from remnant populations9.Habitat degradation and fragmentation result from natural and anthropogenic disturbances, causing the loss of structure that supports entire ecosystems and the services they provide12. Heterogeneity in the responses of ecosystems to large scale disturbances allows remnant, neighbouring populations to supply disturbed reefs where self-seeding is no longer possible due to the loss of the local reproductive population14 (but see ref. 15). In the absence of large environmental fluctuations, coral mass spawning events are closely tied to a lunar cycle and feature synchronised release of gametes by colonies of many species, aided by cues from light, temperature, wind, and tidal fluctuations20. Most colonies spawn in a single synchronised mass spawning event per year or season. But in some years, spawning is split over two consecutive months at the same location24 during which a substantial proportion of colonies of the same species, as well as across multiple species, will spawn in one month or the other. Hence, larval supply can occur more than once per reproductive season during split spawning years.Due to the bipartite life-history of most marine organisms, and corals in particular, understanding larval dispersal and metapopulation connectivity is a central component of reef management27. While the numbers of larvae released during coral mass spawning events are enormous28, dispersal is highly reliant on ocean currents at the time of spawning29. This creates interannual stochasticity in larval supply among reefs, increasing the variability of supply and decreasing the reliability of inter-population connectivity. It has been proposed that the biological mechanism for split spawning is to realign the timing of coral reproduction with optimal environmental conditions24, and may be driven by a disconnect between lunar cues that trigger spawning and environmental cues that drive gametogenesis24. It is also considered feasible, albeit in the absence of evidence, that reproductive success could be impaired during a split spawning event because of lower synchrony that potentially reduces the likelihood of fertilisation success, premature release of gametes (but see ref. 24), or enhanced risk that part of the event misses optimal environmental conditions30. Yet split spawning events could also benefit corals by reducing the temporal uncertainty in larval supply by making supply more consistent over the years, as well as increasing the diversity of source locations that can supply larvae to a reef. In short, some reefs might experience enhanced recruitment owing to the multiple opportunities for supply provided by split spawning.Connectivity in marine systems is inherently stochastic, with interannual variation in both the oceanographic forces and local conditions creating transient links among populations21. The GBR is a complex system of ~3800 interconnected reefs that span more than 2300 kilometres in latitude. Here, we recreate seven years of spawning patterns across the GBR and assess the effects of split spawning events on the spatial and temporal variability, as well as robustness of larval supply to individual reefs. We use oceanographic models to compare the patterns of dispersal of coral larvae with split spawning against hypothetical connectivity patterns that would be obtained if only a single spawning occurred in a year. We find that split spawning events increase the reliability of larval supply by providing a greater number of external larval sources that supply reefs as well as more years during which inter-reef connectivity is likely to occur.Split spawning events do occur on Australia’s Great Barrier Reef (GBR)31. Dispersal of coral larvae was driven by ocean circulation simulations33 with additional parameterisation based on coral larval development from laboratory studies34 and field observations21. To facilitate the interpretation of the results in terms of geographic regions as well as local conditions experienced by the reef35, we divided the reefs latitudinally into north, central and southern sections, and also according to their shelf position into inner-, mid-, and outer-shelf sectors , the number of source reefs that supplied it, and the total number of years (out of a maximum of seven) as well as the period of consecutive years during which a reef was or was not supplied . Relative supply levels and the number of sources were calculated annually to capture both the mean expected value per reef and variability over time.We modelled coral connectivity on the GBR by using ~3800 polygons representing indicative reef boundariesReefs had varying levels of mean annual larval supply Fig. . In geneIf oceanographic conditions were similar between consecutive months of a split spawning event, we would expect the proportion of larvae arriving from the second month to equal that of the relative larval release . Large departures from this proportion imply that the potential for larval supply differs dramatically between consecutive months. For example, if the proportion was 0.1 then 10% of all larvae supplied to the reef stem from the second month of spawning despite the fact that 40% of larvae were released in that month (and 90% of larvae stem from the first spawning month). Great disparity of proportional larval release vs. supply indicates that a single spawning event would have entailed elevated risk of failure. Continuing our hypothetical example, had a single spawning event occurred in the first month then larval supply would have been extraordinarily high because the proportion of supply exceeded release (0.9 vs. 0.6). But had corals spawned only in the second month then supply would have been weak because the reef received relatively few larvae in that month (0.1 vs. 0.4). Our results suggest that absolute levels of larval supply are not particularly sensitive to which month the corals spawn: the GBR-wide average proportion of supply in the second month was 0.36 Fig. , which iReefs in the outer-shelf regions tended to have fewer sources of coral larvae, particularly in the north Fig. . Supply Despite the majority of reefs being regularly supplied during the seven-year period of simulations, half of them (53%) experienced at least one year of failed larval supply from external sources even with split spawning. The vast majority of such failures occurred in mid- and outer-shelf regions Fig. . More thSplit spawning did not only prevent failures of larval supply in single years. Many reefs in outer- and mid-shelf regions would have experienced consecutive years of failure, which in rare cases could reach up to 5 years in the absence of split spawning Fig. . On aver37. A more reliable supply over time from a wider pool of potential sources may be critical for recovery, especially in the early stages after a disturbance impact when populations are in greatest need of larval supply.Coral connectivity on the GBR is highly variable, resulting in large fluctuations in potential larval supply between years. Patterns of connectivity exhibit strong cross-shelf trends with less consistent supply generally a characteristic of outer-shelf reefs. This stochasticity arises from variations in ocean currents and could lead to years with reduced external supply, especially for reefs in outer-shelf regions that appear to have fewer upstream sources. In general, the benefit of split spawning is to approximately double the number of larval sources to each reef and in so doing improve the reliability of larval supply. In the absence of split spawning, 36% of reefs would experience at least one year of failed external supply within seven years. Moreover, the average period of larval supply failure would extend by an average of 0.4 years in the absence of split spawning. Thus, split spawning ameliorates the inherent stochasticity of marine connectivity, and could play a role in enhancing the recovery potential of reefs in regions where connectivity tends to be low and/or highly variable, such as the outer-shelf regions of the northern and central GBR where the combination of oceanography and geography means that upstream sources of larvae are not readily available because currents tend to enter the reef matrix from the open ocean38. In particular, split spawning had a notably positive effect on the diversity of sources that supply a reef, as an additional spawning event within the same year has the potential to provide larvae from a substantially different set of source reefs. This increase in connectivity due to the second spawning event in the same year may increase the resilience of larval supply as more and more potential source reefs become degraded by disturbance impacts.In some areas, strong deviation from the expected proportions of larval release versus subsequent supply indicates that these reefs would receive most of their larvae from only one of the two spawning events in a split spawning. Had corals utilised only a single spawning event the fate of larval supply would be relatively uncertain as the outcome would be highly dependent on the exact month when they spawned. Under these circumstances, split spawning effectively ‘hedges bets’, increasing the chance that at least one of the split events would coincide with favourable oceanographic conditions for larval dispersal and supply. Conversely, reefs that were close to the expected ratio of larval supply were more likely to be supplied equally during split spawning, and therefore had more consistent supply under various oceanographic conditions. Split spawning could still be beneficial to these reefs if, for example, a staggered supply increases the chance that recruits would survive density-dependent early post-settlement bottlenecks24. Yet, split spawning likely involves reproductive costs chiefly because of lower synchrony in gamete release among colonies, which elevates the risk of Allee effects with colonies potentially being too far apart to achieve high levels of successful fertilisation40. An intriguing possibility is that the benefits we describe here — that split spawning may improve the reliability of larval supply — might serve to offset some of the reproductive costs to fertilisation success during such events, as well as other aspects of lower larval concentrations such as reduced swamping of opportunistic predators41. It is too early to model such scenarios because of the absence of data, but continued insights into the biological causation of split spawning42 could enable formal testing of such ideas in future.Mechanistically, split spawning has the benefit of realigning the timing of reproduction with optimal environmental conditions43 that contain ocean flow anomalies44. Constraining the model to just the GBR region may also underrepresent the amount of larvae some reefs would receive from reefs outside the GBR region, e.g., in the Torres Strait where potential sources could be close enough to provide demographically relevant supply. While these edge effects may mean that these reefs might normally receive more supply from external sources and be at a lower risk of supply failure than when considering the GBR alone, they may nevertheless also benefit from split spawning events in the same manner as other reefs.Although the outcomes of this study suggest that most of the GBR would enjoy some benefits in having more reliable supply from split spawning, the period examined was limited to the seven years of data available from the hydrodynamic models. A longer time series might refine these insights further; for example, the southern sector of the GBR only experienced three split spawning events over seven modelled years . Acropora corals were used as a model organism owing to its high abundance and importance as a reef builder and habitat provider63. Acropora are hermaphroditic spawners23 that release egg and sperm bundles in synchrony during mass spawning events into the water column for cross-fertilisation41. Recent meta-analysis has revealed that the major control on the timing of spawning is related to the rapid increase in seawater temperature64, and most corals spawn as waters are warming or close to the annual maxima65. Spawning generally occurs around the transition from spring to summer, peaking 4–6 days following the full moon, but local environmental factors influence timing16 and exact spawning times deviate around this general timing.We simulated larval dispersal along the entire GBR during seven spawning seasons  for which the oceanographic models are available  on each of the predetermined spawning dates. The timing of spawning by latitudinal sector was implemented following the release dates in Supplementary Table Acropora colonies often spawn over a number of consecutive nights, to simplify the simulations we used a single spawning date at any combination of sector x season x month. This resulted in 25 discrete spawning dates  over the course of 7 reproductive seasons for which the oceanographic data are available. Even though the larval depth can change as a function of time due to changes in lipid content67, we presumed an upward swimming behaviour of developed larvae 68. Larvae were therefore driven by near-surface waters (upper metre of the water column) until they reached another reef. Larval competency initially occurs in Acropora ~4–5 days following spawning69, with peak competency generally occurring ~8–12 days after spawning67, followed by a prolonged exponential decline34. Competency and survival curves were incorporated to estimate the abundance of settlement on any given reef, derived using Equations 3 and 4 and Tables 34 for A. millepora, a ubiquitous broadcast spawning coral on the GBR. Location of the particles was recorded in 12-hour intervals from the time of release for a period of 120 days. If at any time larvae were found to be located within a 1 km halo surrounding a reef70, they were considered to contribute to the larval pool of that reef. The amount of larvae a source reef contributed to a destination reef were used to estimate the strength of a directed connectivity link between source-destination pairs. The strength of links were also scaled to the size of source reefs, with the assumption that larger source reefs contribute more larvae and that this contribution is proportional to reef size. While linking the size of a spawning population to reef size does not take into account the possible state of the reef in terms of the relative abundance of live coral on it, this is a necessary assumption in the absence of a comprehensive dataset on coral populations that were, or may have been, present on each individual reef on any given year. The effect of local conditions at a destination reef on post-settlement mortality of Acropora recruits could not be reliably estimated GBR-wide and was therefore not modelled in the present set of simulations, instead assuming that the contribution to the post-settlement population was proportional to the received supply54. As a result, the link strength represented potential, rather than realised, connectivity among reefs.We simulated  evening 0–23 h on21. To standardise the overall amount of larvae released per reef per year in the event of split spawning, we multiplied the strength of the outgoing links for reefs that underwent split spawning by the respective modifier (0.6 for the first spawning of the year and 0.4 for the second one). Finally, we combined the connectivity results for all spawning events during the same year in order to obtain GBR-wide annual connectivity networks.We used contributions of larvae from individual sources to obtain an estimate of larval supply that arrived at a destination reef as a result of a spawning event. Field observations of split spawning on the GBR indicate that ~60% of the colonies spawn during the first month, and the remaining 40% during the subsequent monthTo estimate the potential benefits of split spawning, we compared the relative contributions of the two yearly spawning events to the total connectivity for that year. For this, we assumed that all colonies on a reef would spawn in either the first or the second spawning month of a split spawning year. The difference in connectivity between the two scenarios was then used to determine how connectivity would have changed if a reef underwent only a single spawning event in a year. Supply from reefs in sectors that were not supposed to undergo split spawning in that year were assumed to be unchanged and were added to both values. We made no assumptions about whether an earlier or a later spawning event would have been a more likely outcome had split spawning not occurred. Instead, we examined how the value of connectivity metrics with either single spawning would deviate from the expected values recorded under a split spawning scenario.We used four connectivity metrics to determine the effect of split spawning on potential larval supply to the reef: (1) the relative amount and the coefficient of variation of coral larval supply a reef received from other reefs per year, (2) the number of individual reef sources that supplied it, (3) the total number of years during which it was/was not supplied, and (4) the longest sequence of successive years with no external larval supply over the course of the simulations. To compare larval supply across multiple years, we normalised the amount of supply a reef received per year by dividing it by the maximum annual supply received by any reef during that season, which resulted in values between 0 and 1 for each reef per year. While local retention was present in the simulations and a portion of the larvae would settle on the home reef, thus reducing the number of larvae available for dispersal to other reefs, it had little effect on the presented results given that all of the metrics rely on inter-reef connections , and not on self-connections. This kept the focus of the study on potential for reef-to-reef rescue via larval supply received from external sources. We then calculated per-reef means and coefficients of variation for supply to assess relative levels and temporal variation of annual external supply of coral larvae to reefs. We also compared the amount of external larval supply reefs received during individual spawnings in split spawning scenarios against a null expectation of 60:40 . Reefs with strong deviation from this null ratio were highly dependent on one or the other spawning event for the amount of larvae they received from other reefs in a given split spawning year irrespective of the actual ratio of released larvae.We recorded the mean number of other reefs a reef could expect to have as external sources of larval supply per year, as well as coefficient of variation in the number of these external sources to estimate interannual variability. Since this metric was derived from an unweighted source count, the null expectation here was 50:50, i.e., that the two spawning events in split spawning will contribute the same number of sources to the yearly total. Reefs with strong deviation from this null ratio derived most of their connectivity from a single spawning in a split spawning year, while those that were close to the null expectation enjoyed increased diversity of sources by being supplied by two different sets of sources across the two spawning events in a split spawning year.If a reef did not receive any external supply during a simulated spawning year, that year was counted as an external supply failure for that reef. Reefs with fewer years of external supply failure could, therefore, be considered to have a more reliable reef-to-reef supply of coral larvae even with high variation in interannual connectivity. We calculated the total number of years with external supply failure, as well as the number of successive failure years, for each reef. We also calculated the number of extra years without external supply, as well as the increase in the number of successive failure years a reef would experience without split spawning.31 to better illustrate how split spawning impacts would vary with reef’s position in a seascape.All connectivity outputs were divided into latitudinal as well as cross-shelf sectors see Fig.  using thSupplementary Information


Long-range RNA-RNA contacts mediated by the CRE build the structural scaffold required for its proper functioning. The recruitment of different cellular factors, many related to the functioning of the translation machinery, might aid in the CRE-exerted downregulation of viral translation. The present data show that the CRE promotes a defect in polysome production, and hinders the assembly of the 80S complex, likely through the direct, high affinity recruitment of the 40S ribosomal subunit. This interaction involves the highly conserved 5BSL3.1 and 5BSL3.3 domains of the CRE, and is strictly dependent on RNA-protein contacts, particularly with the ribosomal proteins RPSA and RPS29. These observations support a model in which the CRE-mediated inhibition of viral translation is a multifactorial process defined by the establishment of long-range RNA-RNA interactions between the 5′ and 3′ ends of the viral genome, the sequestration of the 40S subunit by the CRE, and the subsequent stalling of polysome elongation at the 3′ end of the ORF, all governed by the highly stable hairpin domains 5BSL3.1 and 5BSL3.3. The present data thus suggest a new managerial role in HCV translation for these 5BSL3.1 and 5BSL3.3 domains.The RNA genome of the hepatitis C virus (HCV) encodes a single open reading frame (ORF) containing numerous functional elements. Among these, the  In positive, single-stranded RNA viruses, such as the hepatitis C virus (HCV), the genomic RNA acts as a template for both translation and replication. While the ribosomes move along the viral mRNA, viral RNA polymerase must initiate the synthesis of the minus strand, thus creating a potential steric hindrance at the 3′ end of the viral genome. This could render viral protein synthesis inefficient and reduce viral RNA production. Translation and replication must therefore be intimately coupled, and should not overlap in time, demanding the precise control of both.2. The HCV IRES functions by the direct recruitment of the 40S ribosomal subunit at the essential subdomain IIId in the absence of any other factor7. Additional, distant, functional genomic RNA domains enhance HCV protein synthesis9  flanked by highly conserved untranslated regions  has been reported to operate as a dual partner, downregulating HCV protein synthesis11, and enhancing viral replication13. The HCV CRE has been characterised as a set of genotypically conserved structural elements, mapping to the very 3′ end of the ORF, immediately upstream of the translation stop codon16  by promoting the local structural tuning of the 3′ end of the viral RNA genome20, which seems to be essential for replication21; and (ii) by allowing the circularization of the HCV RNA via the formation of long-range RNA-RNA interactions with subdomain IIId of the IRES region23. The acquisition of a closed-loop topology is a crucial step required for both replication and translation; the circular conformation provides nuclease resistance and creates optimal microenvironments in which different processes can occur. Indeed, mRNA circularization is a strategy adopted by most mRNAs to allow the efficient reloading of any ribosome that reaches the stop codon to the 5′ end of the mRNA.The mechanism by which the CRE interferes with HCV protein synthesis is not completely understood. It is known, however, that the 5BSL3.2 domain governs a complex network of contacts that organise the conformation of the HCV genome at two levels24. These observations prompted the proposal that the well-known RNA-RNA interaction network involved in the control of viral translation might be aided via the direct recruitment, or sequestration, of the cellular translation machinery. The present work provides evidence for the direct interaction of the CRE with the 40S ribosomal subunit. This interaction is dependent on RNA-protein contacts, especially with the ribosomal proteins RPSA and RPS29, both of which are involved in the efficiency of IRES-mediated viral protein synthesis26. The present results support a model in which the CRE region sequesters 40S subunits, preventing efficient ribosome reloading at the 5′ end of the mRNA genome, thus repressing translation.Our group has previously isolated ribosomal proteins bound to the CRE regionThe translational competence of an mRNA molecule is related to the efficiency with which it recruits ribosomes. The final number of bound ribosomes depends on the length of the ORF, but it is also influenced by the ease with which translation is initiated, elongation occurs, and termination takes place. Quantification of the association of polysomes with different HCV subgenomic RNA molecules was performed to examine the negative effect of the CRE region on translational efficiency.23  and cellular GAPDH mRNA were chosen to normalise RNA levels and to identify both the non-polysomal and polysomal fractions .The previously reported transcript ICU23 Fig.  was used11. The RNA transcripts derived from the IC molecule with in-phase deletions of the CRE domains - the so-called IC_d3.1, IC_d3.2 and IC_d3.3 transcripts11  in both the affinity and the yield of the reaction compared to those obtained for the CRE region alone . A reaction without the 40S particle was used as a control. The resulting extension products were analysed in high-resolution denaturing polyacrylamide gels with RNA sequencing ladders run alongside to accurately map the cDNA products. The results showed the presence of several specific reverse transcription pauses in the presence of the 40S subunit compared to the extension pattern obtained in its absence nce Fig. , suggest351 Fig. . Taken t27 was next designed to determine the structural requirements at each position for the formation of the CRE:40S complex. For this, the HMX methodology (2′-hydroxyl molecular interference) was followed, using 2′-hydroxyl selective SHAPE chemistry28 with the NMIA (N-methylisatoic anhydride) reagent, as previously described29  modified in the bound RNA (see Methods). In this assay, the CU transcript was used instead of CRE + HV to avoid the loss of information at the 3′ end of the CRE, which would be obfuscated by the outlying fluorescent signal in the shortest extension products.An interference strategyles Fig. . The res9327-U9331, which map to the 5′ flank of the 5BSL3.3 stem. Interestingly, these nucleotides are partially complementary to those mapped by toeprinting at the 3′ side of the 5BSL3.3 stem, confirming 5BSL3.3 as a major binding site for the 40S subunit. From a functional point of view, this finding provides an interesting starting point for investigating the potential role of the 5BSL3.3 domain in HCV translation control.The results revealed that the structural requirements for the establishment of the CU:40S complex rely on discrete regions of the CRE Fig. . Of noteA number of residues in the apical portion of the stem-loop of domain 5BSL3.1 were also identified as critical partners in the formation of the CRE:40S complex, with an average HMX score of 0.95 ± 0.24 . Indeed, several of these nucleotides belonged to the essential bulge of 5BSL3.2  and 29 (RPS29).To determine the ribosomal proteins directly involved, label-transfer assays coupled with a proteomic identification strategy were performed. The ule Fig.  was incuTaken together, these results indicate that the CRE:40S interaction is protein dependent.The CRE region is a highly conserved element of the HCV genome critical for viral translation and replication. How these processes are coordinated and regulated, however, has been difficult to answer. The present work provides evidence that the CRE sequesters 40S ribosomal particles, which might be related to the negative regulation exerted by it on IRES-dependent translation, and the concomitant increase in viral RNA replication.11. It was proposed that the establishment of the long-range interaction IIId-5BSL3.2 might interfere with the proper recruitment of the 40S ribosomal subunit within the HCV IRES region11, resulting in the initiation of protein synthesis being blocked. The results of the present polysome binding assays   at the 3′ end of the HCV ORF33. This observation, together with the finding that the stem of the 5BSL3.3 domain is a major anchoring site for the 40S subunit, suggests an unknown role for 5BSL3.3 as a manager of translation.The anchoring of the 40S subunit to the CRE is dependent on certain ribosomal proteins, among which preliminary analyses include RPSA and RPS29. Like many other ribosomal proteins, RPSA and RPS29 interact with rRNA 18S. Sequence alignment of the CRE and rRNA 18S revealed strong homology between the 5BSL3.1 and 5BSL3.3 domains and the H40 and H41 stem-loops of rRNA 18S Fig. ; the lat31. It recognises the internal asymmetrical loop of the H23 stem-loop of rRNA 18S34, though it seems to show a certain promiscuity with other RNA transcripts that can be targeted by RPS29 and modulated by this protein34. According to the present toeprinting and molecular interference results, the bulge of the 5BSL3.2 domain, and the internal loops of the 5BSL3.1 domain, are feasible targets for RPS29. Studies to confirm the involvement of both RPSA and RPS29 in the formation of the CRE:40S complex are currently underway.RPS29 locates to the interface between the 40S and 60S ribosomal subunits, close to the initiator AUG in the mRNA, following the formation of the 80S translational complex36. The sequestration of these factors by the CRE might be used as a strategy to interfere with viral protein synthesis. This idea is reinforced by the observation that the affinity of the HCV IRES for RPSA-deficient 40S subunits is reduced37, providing firm support for the idea that the recruitment of RPSA helps regulate HCV IRES-dependent translation.It is noteworthy that both RPSA and RPS29 have been identified in HCV IRES-bound complexes by following different proteomic strategies39. RPS29, in contrast, enhances the tumour suppressor activity of the Ras-related protein 1A40. These functions provide clues about the theoretical consequences that the sequestering of RPSA and RPS29 by the CRE might entail, such as avoiding co-infection with other members of the Flaviviridae family or the induction of hepatocarcinoma. This points to new roles of the CRE region beyond the control of viral translation and replication.In addition to their roles as ribosomal components, both RPSA and RPS29 act as soluble or receptor proteins in the cell. In this context, RPSA is a laminin-binding protein with implications in tumour invasion. It can also operate as a potential receptor for the envelope proteins of different flaviviruses, such as West Nile or Dengue virus11, but also 5BSL3.1 and 5BSL3.3. Three different regulatory pathways could define such an inhibitory mechanism: (i) first, the interaction IIId-5BSL3.2 would impair HCV IRES-dependent translation by impeding the recruitment of the 40S subunit by subdomain IIId11; (ii) second, the high affinity binding of the 40S subunit to the CRE region, either at the 5BSL3.1 or the 5BSL3.3 domains, or both, might prevent active 80S ribosomes from penetrating the 3′ end of the viral ORF. This might promote the stalling of polysome production and impede the proper re-loading of the 40S subunit at the 5′ end mediated by the 3′UTR10. This defect in ribosome penetration would be enhanced by the presence of highly stable hairpins (formed by 5BSL3.1 and 5BSL3.3) that cannot be bypassed by the 80S translational complex41. A major consequence would be an intracellular increase in inefficiently translated viral RNA genomes, but which could be used as replication templates; iii) finally, the sequestering of translation factors by the CRE might be considered a feasible means of interfering with HCV protein synthesis. Such a hypothesis is supported by previous reports describing the binding of the translation machinery proteins to the CRE region24. Further work is needed to clarify whether these in vitro findings can be exported to an ex vivo infection model.Taken together, the present results support a working model in which the translation inhibition effect mediated by the CRE can be envisioned as a multifactorial process involving not only the 5BSL3.2 domain as previously proposedIn summary, the present work identifies a high affinity interaction mediated by the CRE and the 40S ribosomal subunit, confirming the multivalent role of the CRE region as a regulator of replication and as a recruiting centre for the translation machinery. The proposed interaction supports the idea that CRE-mediated translation is a multifactorial process involving different capacities and functionalities of the CRE, and suggests new and unsuspected roles for 5BSL3.1 and 5BSL3.3 as switching partners that manage the transitions between the different steps of the HCV cycle.24.The DNA template constructs for IC, ICU, IC_d3.1, IC_d3.2, IC_d3.3, C, CRE + HV, U, I + CU and CU RNA were obtained by amplification using specific primers as previously describedXbaI. RNA667 was obtained as previously reported22.The template for RNA-100 was generated by endonuclease digestion of the pBS SK(+) plasmid with in vitro synthesised using the TranscriptAid T7 High Yield Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. The quantity of RNA produced was determined by UV spectrophotometry (A260); protein and carbohydrate/phenolic contamination was assessed via the A260/A280 and A260/A230 ratios respectively. The integrity of the transcripts was tested by denaturing agarose gel electrophoresis. Internally radiolabelled RNA molecules were synthesised as previously described22.RNA molecules were 2 atmosphere.Human hepatoma Huh-7 cell monolayers were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum (Invitrogen) and 1 mM sodium pyruvate (Sigma), at 37 °C in a 5% CO11. Briefly, 48 h before transfection, 500,000 Huh-7 cells were seeded onto 10 cm-diameter plates to reach 90% confluence. 5 µg of the constructs IC, ICU, IC_d3.1, IC-d3.2 or IC_d3.3 and 1 µg of the RNA667 were then mixed with 50 µl of Opti-MEM® (Invitrogen) and 2 µl of transfection reagent  and added to cell cultures. Translational efficiency was analysed by polysome profiling.Cell transfections were performed essentially as previously reported42. Briefly, S10 fractions from ~4 g of Huh-7 cells were prepared as described24. Ribosomes were then loaded onto 3 ml of a 1 M sucrose cushion in buffer A  and precipitated by ultracentrifugation at 40,000 rpm and 4 °C in a Beckman 70.1 Ti rotor for 4 h. The resulting pellet was resuspended in buffer B  to a concentration of 50–150 A260 units. This suspension was incubated with 4 mM puromycin for 10 min at 4 °C, following by incubation at 37 °C for 30 min. Salt concentrations were then increased by the addition of 0.5 M KCl to release the translation initiation factors bound to the ribosomal subunits. The ribosomal subunits were then loaded onto a continuous 10–45% linear sucrose gradient in buffer A and resolved by ultracentrifugation in a Beckman SW40 rotor device at 28,000 rpm overnight. Fractions of 500 µl were collected from the top of the gradient and concentrated using Amicon® Ultra-2 30 kDa. The concentration of the 40S subunits was calculated by UV spectrometry measurements as 1 A260 = 30 pmol/ml.40S ribosomal subunits were isolated from freshly prepared Huh-7 S10 cell lysates as previously reported, with slight modificationsg for 5 min at 4 °C, and the pellet lysed in 2 volumes of polysome lysis buffer . Cell lysate was centrifuged at 11,000 × g for 10 min at 4 °C, to pellet nuclei and debris. The supernatant was overlaid onto a linear 10–50% sucrose gradient in buffer A . Gradients were centrifuged for 2 h at 190,000 × g in a Beckman SW40 swinging bucket rotor apparatus at 4 °C. 1 ml fractions were collected from the top of each gradient and supplemented with a fixed amount (100 ng) of the non-related and non-translatable RNA667 transcript, used as internal control for the subsequent RT-qPCR steps. Fractions were then subjected to RNA extraction with TRIzol reagent (Thermo Fisher Scientific), following the manufacturer’s instructions. RNA integrity was monitored by gel electrophoresis in a 1% agarose gel and quantified by densitometry. The fractions showing a 28S/18S ratio of ≥1.6 corresponded to those bearing polysome-bound RNA. The distribution of the specific RNA molecules containing the HCV subgenomic constructs was determined by RT-qPCR and normalised to that obtained for the GAPDH mRNA, essentially as previously described43 with minor modifications. RT-qPCR was additionally performed for the RNA667 and referred to that obtained for the GAPDH mRNA, with the aim of corroborating that RNA extraction and precipitation processes accounted with similar efficiency for all the fractions tested. Briefly, 500 ng of RNA were hybridised with 3 µg of random primers by denaturation at 95 °C and cooling on ice. MultiScribe reverse-transcriptase was then added to a final concentration of 0.5 U/μl and cDNA synthesis performed in the presence of dNTPs (0.5 mM) and the buffer supplied by the manufacturer. The extension proceeded in two steps: slow extension at 16 °C for 30 min, followed by rapid extension at 37 °C for 30 min. The reverse transcriptase was heat-inactivated at 85 °C for 5 min. One sixth of the cDNA mix volume was employed for qPCR with the SsoFast™ Evagreen® supermix (Bio-Rad) and amplified over 40 cycles with specific oligonucleotides targeting the IRES region (C-149 and C-342)44 or the RNA66724.Transfected Huh-7 cells were treated with cycloheximide (0.1 mg/ml from Sigma-Aldrich) at 37 °C for 20 min and then washed twice with PBS supplemented with cycloheximide (0.1 mg/ml). The cells were scraped off and resuspended in ice-cold PBS in the presence cycloheximide (0.1 mg/ml). This suspension was centrifuged at 800 × d) for the HCV RNA:40S subunit. For this, the 32P-internally radiolabelled RNA molecules C, CU, U and I + CU were heated at 95 °C for 2 min and then cooled to 4 °C. A concentration of 0.1 nM of the RNA constructs were then incubated with increasing concentrations of the purified 40S subunits (0–100 nM) in binding buffer , 100 mM; KCl, 200 mM; Mg(AcO)2, 2.5 mM; DTT, 1 mM) for 30 min at 37 °C. The reactions were then loaded onto a filter sandwich composed of 0.45 µm nitrocellulose filters  able to retain the ribonucleoprotein complexes; these were placed above nylon filters  to trap any RNA not captured by the nitrocellulose membrane. Both nylon and nitrocellulose membranes were pre-soaked in binding buffer and assembled in a dot-blot apparatus. Samples were diluted up to 100 µl with binding buffer and were directly added to the filters under vacuum. Filters were then dried and scanned in a Phosphorimager  and quantified with Image Quant 5.2© software . Kd values were calculated using Sigma Plot 8.02© software according to the equation y = y0 + (Bmax·xn)/(Kdn + xn), where y is the percentage of complexed RNA, Bmax is the binding yield, x is the concentration of the target RNA, Kd is the dissociation constant and n is the Hill coefficient.Binding assays were performed with the aim of calculating the dissociation constant values (K32P-end labelled oligonucleotide P422 (2 pmol) was denatured and refolded by heating at 95 °C for 2 min followed by slow cooling to 37 °C for 15 min to minimise non-specific interactions. Purified 40S subunits (200 nM) were then added to the mixture and binding reactions allowed to proceed in the presence of binding buffer for 30 min at 37 °C. The control reaction involved substituting the 40S subunit for an equivalent amount of a non-related RNA (RNA667). For subsequent reverse transcription, of SuperScript III reverse transcriptase (200 U from Thermo Fisher Scientific) and dNTPs (0.5 mM) were incubated in a 20 µl reaction volume with the reverse transcriptase buffer provided by the supplier. Extension was allowed to proceed for 30 min at 37 °C and stopped by snap cooling on ice. The reaction products were resolved by high-resolution denaturing polyacrylamide gel electrophoresis (4–6%), dried, scanned in a Phosphorimager , and quantified using Image Quant 5.2© software .For each 10 µl reaction volume, a mixture containing the CU RNA (1 pmol) and the 29, with minor modifications. Briefly, the RNA construct CU (5 pmol) was subjected to chemical modification under denaturing conditions (heating at 95 °C) in the presence of 100 mM Hepes pH 8.0 and 25 mM of the modifying reagent NMIA [N-methylisatoic anhydride] for 3 min). The reactions were then cooled on ice and the modification protocol repeated twice. The final reaction volume (20 µl) was achieved at every modification step by replacing the evaporated water. The modified RNA was ethanol-precipitated and the final quantity monitored by UV spectrophotometry (A260). Complex formation between the CU molecule and the 40S subunit was accomplished at a molar ratio of 1:5 in binding buffer for 30 min at 37 °C. This complexed RNA-CU was separated from the unbound CU molecules by differential filter retention assays, as described above. RNA molecules were passively eluted from the filters for 3 h at 4 °C and subjected to phenol:chlorofom extraction and ethanol precipitation. NMIA modifications were detected by primer extension and the cDNA products analysed by capillary electrophoresis, as previously described20. HMX scores were calculated as previously reported29.HMX analysis was performed essentially as previously described32P labelled RNA C was incubated with a 5-fold molar excess of the purified 40S subunit in cross-linking buffer , at 37 °C for 30 min. In parallel, C:40S complex formation was completed with a 5-fold molar excess of the non-labelled RNA C. Control association reactions were also performed with RNA-100, a non-related RNA that does not bind the 40S subunit. Reactions were then exposed to 365 nm UV light for 15 min using a CL-1000 Ultraviolet Crosslinker (UVP). Samples were placed in a cool metal block to prevent them from overheating. The RNA-protein interaction was verified by adding 1 µg of proteinase K in 0.5% SDS and further incubation for 30 min at 37 °C. Samples were resolved in 6% denaturing polyacrylamide gels, which were dried and scanned as previously described45.RNA denaturation and complex formation were performed under similar conditions to those described for the binding assays. Briefly, 10 nM of the internally To identify those ribosomal proteins directly targeting the CRE RNA, the cross-linked binding reactions were treated with RNase A 50 ng/µl and loaded on 14% denaturing SDS polyacrylamide electrophoresis gels. Products corresponding to every RNA-protein complex were cut out and digested with trypsin overnight. Peptides were purified in 0.2% TFA and 30% acetonitrile, crystallised in an “AnchorChip” plate using 0.7 mg/ml CHCA as a matrix, and analysed in an UltraXtreme mass spectrometry MALDI-TOF/TOF device (Bruker). Proteins were identified using the MASCOT search engine, checking against the Swiss-Prot database and allowing for 1–2 trypsin missed cleavages. Only proteins with a Mascot Score of over −10xLog(P) were contemplated (where P is the probability that the observed match is a random event).Supplementary Information


Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding.Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats ( Four families of viruses that are pathogenic for other mammalian species  may also have originated in bats. These viruses often cause serious disease in their secondary hosts, but most do not appear to cause clinical signs or pathology in bats18, suggesting that uniquely benign relationships have co-evolved between the viruses and their primary bat hosts20. While relatively little is known about the dynamics of viral infections in bats, these viruses may be maintained in bat populations as a result of either persistently infected individuals, reinfection after waning immunity, or spatial transmission dynamics22.Bats are hosts for many viruses and are thought to be the source of some viruses that have spilled over to humans and other mammals, causing fatal disease. These include coronaviruses causing severe acute respiratory syndrome , big brown bat (Eptesicus fuscus), and northern long-eared bat . All three species hibernate from October to May, sometimes in shared hibernacula. We recently demonstrated that ~30% of hibernating M. lucifugus sampled over two years from hibernacula in Manitoba were infected with a coronavirus (Myl-CoV), which persisted at low levels in the intestine32. A closely related coronavirus also infects E. fuscus33.The Canadian prairies are home to three species of bats, including the little brown bat (37. The fungus (Pseudogymnoascus destructans) causes white-nose syndrome (WNS) in hibernating bats, which is characterized by the growth of white fungal mycelia on the face and exposed skin of the wings and tail membranes. The visual and microscopic effects of P. destructans on the skin of the wings are associated with increased expression of several genes devoted to innate immunity and inflammation in wing tissue39. Profound systemic effects include dehydration, hypovolemia, metabolic acidosis, and fat depletion, which can lead to death42. Other systemic effects of bat WNS include an accumulation of neutrophils in the lungs, which is accompanied by an increase in the expression of several cytokine genes43 suggesting that even the most severely afflicted hibernating bats are capable of at least some systemic immune response to fungal infection.Whereas bats appear to be relatively resistant to viral infections, a cold-adapted fungus that was recently introduced to North America has caused widespread mortality in some species of bats in eastern United States and Canada45. Similar interactive impacts of co-infection with P. destructans and viruses on bat immune responses are not known. We used M. lucifugus experimentally-infected with P. destructans and/or naturally infected with Myl-CoV as a model to understand how co-infections influence bat-virus interactions. This system allows us to avoid confounding factors of direct pathogen-pathogen interactions, because the fungus affects the skin, while the coronavirus infections occur internally, almost exclusively in the ileum and lungs32. We hypothesized that co-infection would alter the molecular response of bats to a persistent viral infection, and that viral shedding would change as a result of the increased or disrupted host immune response. To test this prediction, we examined tissues collected from M. lucifugus at the termination of an earlier study that quantified the effects and pathogenesis of P. destructans in hibernating bats experimentally infected with the fungus37, some of which were naturally infected with Myl-CoV32. This combination of uninfected, virus-infected, fungus-infected and co-infected M. lucifugus allowed us to test our hypothesis that host responses to co-infection are synergistic and not simply additive.Previous studies on other species have demonstrated that a fungus and a virus could interact during co-infection and affect each otherMyl-CoV and M. lucifugus RNA through reverse transcription quantitative PCR (RT-qPCR) and dual-RNA-sequencing indicated that co-infected bats had significantly higher levels of Myl-CoV RNA than bats infected with virus alone. The amount of Myl-CoV RNA correlated with the severity of WNS pathology in co-infected bats. This phenomenon was associated with specific molecular responses to co-infection, even in the intestines of bats where only one of the two pathogens was directly interacting with the host tissue. The levels of antibodies against Myl-CoV nucleocapsid (N) protein were also higher in co-infected bats. Each key result is discussed in detail below.Quantitation of Myl-CoV genomic RNA was detected in bats infected only with Myl-CoV , co-infected bats (European P. destructans (3/13), or with North American P. destructans (7/16)37). There was no difference in the frequency of Myl-CoV detected among these treatments . We pooled bats infected with the two P. destructans isolates for all further analyses and tested whether co-infection with P. destructans and Myl-CoV correlated with an increase in viral replication. Our RT-qPCR data showed that the co-infected bats contained 60-fold more Myl-CoV RNA on average than the virus-infected bats , compared to the relative quantities of Myl-CoV RNA in the co-infected bats  and all the bats with WNS into the other group   Fig. .Figure 3M. lucifugus to P. destructans as described in Warnecke et al.37. We performed IgG ELISA on blood plasma to detect Myl-CoV (coronavirus) N protein antibodies and found that 21/63 were positive for antibodies against the coronavirus. Of those 21 bats, 7 had detectable coronavirus RNA in their intestines suggesting an active infection, and 3 out of the 7 had been experimentally infected with P. destructans during the course of the study. We compared the ELISA optical density (O.D.) values of these virus-infected bats to co-infected bats , antigenic drift of virus (e.g. influenza virus) or waning immunity  that allows reinfection, and long-lasting latent (e.g. herpesviruses) or persistent infections (e.g. pestiviruses) with sustained or periodic shedding. It is not yet clear how bat viruses are maintained in their natural host populations, or how they avoid extinction as host populations become immune and less susceptible. Persistent infections can be established in cultured cells with viruses that may have originated in bats, including Ebola virusM. lucifugus in controlled laboratory hibernation chambers for four months during these experiments, and we detected the coronavirus i.e. Myl-CoV, at the end of hibernation. These data imply that the coronavirus can persist in its host for at least the duration of hibernation, particularly as nucleotide variability among the detected coronavirus isolates showed that spread of coronavirus among bats within a chamber was unlikely32. In an extensive study of New World alphacoronaviruses, no target viruses were detected in the rectal swabs of individual bats sampled over time46, suggesting that persistence and intensity of shedding varies among species or viruses. The authors concluded that the targeted coronaviruses do not persist in their hosts but are maintained in populations by the introduction of new susceptible individuals. However, their results could also reflect viral persistence in individual animals, with low baseline levels of virus replication and undetectable shedding interspersed with periods of increased replication and shedding that did not occur during the sampling period.We maintained 29 suggest persistent or latent viruses may be activated by hormonal or other systemic cues. Direct evidence linking a specific trigger to increased shedding has not yet been found. However, viral replication in rodent and bat cells persistently infected with Ebola virus increased greatly following modulation of the Ras/MAPK pathway with lipopolysaccharides or phorbol esters, and with the resulting suppression of the cells’ interferon response49. In experimental systems, the immune modulatory cytokine IL10 also influences viral persistence and replication53, although more study is required to clarify the effects of circulating cytokines on the replication of persistently infecting viruses. Nevertheless, these results suggest that circumstances which induce anti-inflammatory cytokines or suppress anti-viral innate responses, may provide a trigger for increased shedding of persistently infecting virus.Periodic or seasonal increases in virus shedding associated with parturition, lactation, nutritional deprivation or environmental stressp value = 0.07). IL22RA1 is the receptor present on host cells, including intestinal cells, which help in initiating cellular signalling in response to IL22 produced by T-cells54. IL-22 leads to an increase in anti-microbial peptide production, cellular protection against damage and increases cellular proliferation55. Therefore, reduced IL-22 signalling in the intestines of bats with WNS, might suppress the bat defences that control the coronavirus infection. Additionally, previous studies have shown that the anti-inflammatory gene, IL10, is expressed more in the lungs of bats with WNS than in bats without it43. We saw a similar trend with the levels of IL10 in the intestines which might play a role in suppressing the immune response against the coronavirus. Another altered cytokine gene which was of interest was the suppressor of cytokine signalling-6 (SOCS6) gene. Fungal-infected bats showed lower levels of SOCS6 transcripts, lack of which has been implicated in mild growth retardation in mice56. Overall, our results suggest that WNS triggers changes in gene expression in the ileum  replication. In addition to interferon-related genes, we also found that RERG, which is related to growth inhibition, was upregulated in the fungus-infected bats when compared to virus-infected bats. Upregulation of RERG could affect the rate of cell proliferation in the intestines59. Finally, this cascade of responses is associated with increased severity of WNS symptoms.We discovered that bats with WNS (fungus-infected and co-infected) had significantly lower intestinal levels of transcripts for IL22RA1 and other interferon-related genes as compared to uninfected bats, and we observed the same trend in IL10 . Direct interactions between the host and the secondary pathogen are limited to the affected tissue, but the systemic response to the disease triggers a cascade of immune responses, including increased release of cytokines or neutrophils. Affected cells such as intra-alveolar macrophages in the lungs or cells lining the intestine, may produce pro- or anti-inflammatory molecules and influence cells that harbour viral genomes. This cascade of host responses disrupts the equilibrium between the persistently infecting virus and the cell’s innate immune response, leading to a dramatic increase in the expression of coronavirus (Myl-CoV) replication.Based on our results, we propose a model for how secondary infections may increase the replication and subsequent shedding of persistently infecting virus Fig. . The estOur assays were unfortunately limited to analysing viral and cytokine transcripts rather than protein, because reagents for detecting bat viral and host proteins are not yet available. We were not able to perform serial dilutions of the plasma to precisely quantify anti-viral titre due to the limitation in the amount of plasma obtained from each bat. The sample size was also small for this assay, because only 7 of the sampled bats had detectable levels of coronavirus in their intestines and were positive for viral antibodies. Despite these limitations, we demonstrated higher antibodies against the coronavirus in the plasma of co-infected bats when compared with virus-infected bats. This increased antibody level in co-infected bats might reflect an adaptive immune response to increased coronavirus replication in the intestines.35. Variation in the microclimate, and other ecological factors may drive some of this variation60, but our data suggest that cryptic viral infections may also play a role in determining survival rates for bats hibernating in sites colonized by P. destructans. We recommend that future studies on population-wide impact of WNS incorporate viral sampling to help better understand the role of co-infections on bat populations in the wild.Our proposed hypothesis for the mechanism driving increased viral replication following pathogenic co-infection was worth testing, but our results are also consistent with an alternative hypothesis. Increased viral replication or viral load may affect the severity and population-level impacts of WNS. Bat mortality following the arrival of WNS varies widely from site to site, with populations decreasing from 30% to 99%M. lucifugus were collected from a WNS-free cave in Manitoba, Canada in November 2010. Details of the experimental design as well as protocols for collecting and transporting bats, infection with P. destructans, maintenance of bats in hibernation and sample collection have been described previously43. Briefly, bats in groups of 18 were either sham-inoculated or inoculated with North American or European isolates of P. destructans. Bats were housed at 7 °C and >97% relative humidity with ad libitum water. All bats were equipped with data loggers to monitor skin temperatures. Bats were euthanized during the experiment when humanely required or at the termination of the experiment 120 days after inoculation. Immediately following euthanasia samples from segments of wing as well as various tissues were preserved in RNAlater  or in formalin. Samples in RNAlater were kept at −20 °C until they were processed. North American and European isolates of P. destructans caused similar disease outcomes37, so we did not differentiate between the strains in subsequent analysis. The procedures for care, handling and euthanasia of bats were approved by the University Committee on Animal Care and Supply of the University of Saskatchewan (Protocol #20100120). Bats were collected under the province of Manitoba Wildlife Scientific Permit WB11145.Fifty-four male M. lucifugus were collected from a WNS-free cave in Manitoba, Canada in January under the Manitoba Sustainable Development Wildlife Scientific Permit No. SAR16009. Bats were euthanized during the experiment when humanely required or at the termination of the experiment 70 days after infection and a similar experiment was performed at the University of Winnipeg as described above (Protocol #AE08399).In 2017, a further 129 During necropsy, we collected representative samples for histopathology from all major organ systems. In addition, representative samples were taken from all areas of the wing and rolled on dental wax before placing in 10% neutral buffered formalin. Tissues were processed routinely for histology. Five µm sections were cut and stained with periodic acid-Schiff stain to highlight fungal hyphae. Liver and other tissues were processed routinely and stained with hematoxylin and eosin. Wings were scored on a scale of 0 to 5 with 5 being very severe with >50% of wing covered in fungal hyphae. We used a bacterial score from 0 to 5, with 5 indicating wide-spread and abundant bacteria being present in many areas within the dermis and underlying connective tissues. Average scores from 5 sections of wing were used for analysis. Interstitial lung neutrophil assessment was similarly evaluated on a scale of 0 to 5, with 5 being very severe. Average scores from the 5 sections were used for analysis.Tissues preserved in RNAlater were homogenized in 2 ml sealed vials with a 5 mm stainless steel bead, 0.1 g of 0.1 mm zirconia/silica beads and 350 μl Buffer RLT Plus  using a Retsch MM400 Oscillating Mill at 30 Hz for 4 min. Total RNA was extracted following the manufacturers protocol. RNA integrity was assessed using RNA 6000 Nano Kit  with the Agilent 2100 Bioanalyzer.cDNA was synthesized from 1 μg of RNA (or less if concentrations were too low) per reaction using QuantiTect Reverse Transcription Kit (Qiagen 205313). cDNA samples were stored at −80 °C until they were used for PCR.Myl-CoV. Primers were designed from the partial sequence of Rocky Mountain bat coronavirus replicase (accession number EF544563)  Table . The pri43. As there were no differences in results, all subsequent experiments used only GAPDH as a normalizer using primers GAPDH US and GAPDH DS . We quantified coronavirus with RNA primers MyCVF2 and MyCV R1 Table . For iniM. lucifugus genes: c-jun (Accession number: XM_006096110.1), cyclin D1 (XM_006098046.1), IL10 (XM_006094865.1) and TNF alpha (XM_006104644.1). The interferon beta primers were designed using the annotated transcript for the E. fuscus gene (XM_008145044.1), which also amplify transcripts from M. lucifugus. Primer efficiencies were determined from cycle threshold (Ct) values of purified PCR products serially diluted and re-amplified. Primers amplified targets with an efficiency of about 100% and in all cases the identities of the PCR products were confirmed by their specific dissociation temperature, specific sizes on agarose gels and by sequencing.To verify the RNAseq data, cDNA from ileum samples in which coronavirus RNA had been detected via RT-qPCR were analysed using the following primers, IL22RA1, IRF1, RERG and SRC . The thermal profile used was 95 °C for 5 min followed by 40 cycles of 95 °C for 10 sec, 60 °C for 30 sec (readings taken at 80 °C), and a final cycle of dissociation of product 95 °C for 1 min, 55 °C for 30 sec and 95 °C for 30 sec (readings taken at every degree between 55 °C and 95 °C). Only results from reactions that yielded unambiguous results were used for analysis.Myl-CoV is present in detectable concentrations32. Extraction of RNA from ileum tissue, which includes the ileum and potential gut contents have been described in previous sections. Bats were screened for Myl-CoV using RT-qPCR, and bats were assigned post hoc to treatment groups representing four infection histories , 3) Fungus-infected , or 4) Co-infected . All samples had adequate RNA quality for sequencing .To explore the mechanisms driving high virus load in bats with WNS, we performed RNA-seq analysis which could potentially screen all targets in the bat intestinal cells. We targeted the ileum transcriptome because this is the tissue in which ies Fig. : 1) UninTissues were homogenized in 2 ml sealed vials with a 5 mm steel bead, 0.1 g of 0.1 mm zirconium silica beads, 350 µL of RLT buffer (with β-mercaptoethanol)  using a Retsch MM400 tissue homogenizer at 30 Hz twice for 2 minutes each. Total RNA from tissues was extracted using the procedure provided with the RNeasy Plus Kit.Total RNA was sent to The Centre for Applied Genomics at The Hospital for Sick Children . RNA quality was assessed using a Bioanalyzer (Agilent Technologies). We retained all samples with a DV200 (percentage of RNA fragments greater than 200 nt) greater than 85% Table , discard61 to assess sequence quality and Trimmomatic v0.3662 to remove the adapter sequences and low-quality bases from reads with the following settings: Illumina clop:2:30:10, leading:3, tailing:3, slidingwindow:4:15, minlength:36. We used TopHat v2.1.163 to align the trimmed paired-end reads from each library, separately, to the Ensembl M. lucifugus genome sequence (Myoluc2.064) in strand-specific mode (fr-firststrand) with mate-inner-dist values specific for the insert size of each library. We used featureCounts65 to count reads mapped to the Myoluc2.0 genome annotation in strand-specific mode (reversely stranded), counting paired-end reads as fragments, counting only those fragments where both reads aligned successfully, counting multi-mapping fragments, and excluding chimeric fragments. We assessed the variability within and between the treatments using the R package SARTools v.1.3.066. The featureCount-estimated gene counts were transformed by a variance stabilizing method (VST) using SARTools.We used FastQC v0.11.5p-value adjustment method), and locfunc = median (estimate size factors). Differentially expressed genes were identified as having a fold-change >2 and false discovery rate (FDR)-corrected p-values < 0.0567. We produced volcano plots representing the differential expression comparisons by plotting the log of the adjusted p value as a function of the log ratio of differential expression. We used the Ensembl gene IDs identified by DESeq2 as input for the web-based g:Profiler68 to test for gene ontology (GO) term enrichment among the differentially expressed genes, using a FDR significance threshold <0.05. These GO-terms and their corresponding p-values were used in REViGO69 to visualize significant enrichment of biological processes.We identified differentially expressed genes between each of the treatments using DESeq2 v.1.12.3, run in SARTools. Custom SARTools-based DESeq settings included: cooksCutoff = TRUE (perform outliers detection), independentFiltering = TRUE, alpha = 0.05 , pAdjustMethod = BH  in a total volume of 100 µl. Plates were covered and incubated overnight at 4 °C and washed three times with 300 µl of PBS-Tween 20 (0.1%) immediately prior to use. Serum samples were diluted to 1:100 in PBS-Tween 20 (0.2%) supplemented with 5% fetal bovine serum . 100 µl of each sample was added in parallel to a positive and negative antigen plate and incubated at 37 °C for one hour and washed as above. A peroxidase-labelled goat anti-bat IgG secondary antibody  was added, incubated for one hour at 37 °C and washed as above. Peroxidase substrate ) was added to each well and colour development was quantified 30 minutes later by measuring the optical density at 405 nm using an ELISA microplate reader. The ELISA cut-off value (0.39) was calculated as the [ + ].Purified, glutathione-s-transferase (GST)-tagged Data from RT-qPCR and histopathological scores were analysed with SPSS Statistics version 23. The relative levels of a transcript for each bat were calculated as RT-qPCR Cycle threshold (Ct) normalized separately (ΔCt) to the “house-keeping” gene GAPDH. A ΔCt reduction of one (1) indicates an approximately two-fold higher concentration of RNA. The significance of differences of mean values of ΔCt between co-infected bats and virus-infected bats were determined using an independent-samples Mann-Whitney U test. We calculated Pearson’s coefficients to test the correlation between ΔCt levels for coronavirus polymerase cDNA for bats in each treatment class, and average scores for fungal hyphae, secondary bacteria, oedema, necrosis and inflammation in wing lesions, as well as bacteremia and levels of neutrophils in lung, spleen and liver interstitium.Bat studies were carried out in strict compliance with Canadian Council on Animal Care guidelines and the procedure for care, handling, and euthanasia of bats were approved by the University Committee on Animal Care and Supply of the University of Saskatchewan (protocol s#20100120).Supplementary Fig 1Datasets S1-S9


We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. To this end, we added an oligonucleotide to the 5’-end of RNA fragments—a 5’-marker—to mark the read-through cDNAs. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins’ RNA-binding sites. Thus, cDNA start sites confidently identify a protein’s RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5’-marker. As part of the individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) protocol, crosslinked protein–RNA complexes are immunopurified and the RNA fragments are released by protein digestion, resulting in RNAs with a covalently bound peptide at the crosslink site . This isIn iCLIP, the start of the truncated cDNAs should be equivalent to the position of the crosslink sites. This also applies to related techniques that rely on analysis of truncated cDNAs, including among others the FAST-iCLIP, CITS-CLIP, BrdU-CLIP, irCLIP, eCLIP and miCLIP, as reviewed recently . Using t2 254 nm UV light. The cell lysate was prepared and the immunoprecipitation performed as previously described (The modified iCLIP protocol is based on the previously described protocol with modifications that enable the definition of read–through cDNAs (https://github.com/jernejule/non-coinciding_cDNA_starts).The scripts used for the analyses in this paper are available in a fully documented format at the GitHub repository  and the pre-existing eIF4A3 dataset  after collapsing cDNAs with the same UMI and the same starting position to a single cDNA.Unique molecular identifiers (UMIs) were used to remove cDNAs that are a product of PCR amplification  of reads correspond to truncated cDNAs. The authors also identify an important caveat to their work- the quantification of read through cDNAs depends on the ligation efficiency of the 5’RNA oligo. In other words, it is possible that the truncated cDNAs are contaminated with unligated readthrough products. One possible improvement could be to perform an affinity selection of the ligated RNA products, using the 5’ oligo as a handle, prior to cDNA synthesis. In this case, all of the RNA molecules would contain a marker. Any reads lacking the marker sequences could only arise from truncation. We also suggest the authors highlight the extent to which their truncation percentages could vary with ligation efficiency. In summary, this is a technically sound experiment with literal interpretations of the data. The authors include sufficient experimental detail to reproduce the study. Minor point: In the results section, when describing the nucleotide composition at the first position of the read through and truncated , the authors identify the wrong panels. Figure 1b,d correspond to read through and c,e correspond to the truncated cDNA:We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Huppert and Haberman et al. presenta revised version of the widely used iCLIP protocol called “read-through marking” that is able to differentiate read-through cDNAs from the truncated ones that result from the stoppage that occurs when the reverse transcriptase encounters the covalent adduct of RBP-RNA complex at the site of UV-crosslinking site during reverse transcription in the process of making the cDNA library. This method involves the ligation of a 5-end oligonucleotide to the RNA fragments and thus distinctively marks the cDNAs that experience read-through during reverse transcription. Overall, this method is a valuable addition to the CLIP technology and will contribute to increasing the resolution of RBP binding site detection by workers in the field. Since the invention of the original HITS-CLIP and iCLIP methods, that offer detection of the exact RBP binding site and RNA-protein crosslinking site in the target RNAs at single nucleotide resolution, multiple variations of the technology have been developed, such as fast-iCLIP, irCLIP and eCLIP. However, the majority of coordinated computational analysis tools and mapping pipelines for these technologies do not utilize the abundant information provided by these technologies that give the precise location of RBP-RNA binding. This is largely due to the fact that a  portion of the resulting cDNAs are read-through events and do not record the exact crosslinking site of RBPs to the RNA target. As a result, computational analyses suffer from noise introduced by the read-through events that are almost impossible to differentiate from the actual truncated cDNAs. Huppert and Haberman et al., on the other hand, provide an avenue to solve this problem using a straightforward experimental measurement from the technology itself and the data presented in the manuscript strongly support the validity of their new method. We believe the field will benefit greatly from this method and analytical tool, especially in providing increased precision  and detection of RBP binding sites. We only have minor comments about the manuscript:Can the authors specify further about the cDNA start site identification pipeline?Is any peak calling involved? Or are the results presented in Figures 1b-e coming from analyzing the raw cDNA start sites from all cDNA fragments?  Can the authors comment on situations when the reverse transcriptase does not truncate exactly at the crosslinking site, but instead truncates after the crosslinking site, but before reaching the 5’-end of the RNA fragment, in which case this modified analysis software will not be able to catch these events?  We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Here the authors describe an approach to quantify the degree of reverse transcriptase read-through during iCLIP cDNA library preparation. By ligating a specific oligonucleotide to the 5’ ends of cross-linked RNAs, they can roughly quantify the degree of read-through and determine whether the sequence characteristics of the read-through fragments are similar to the truncated cDNAs. The approach used is clever and the paper is of a good quality. I do have a few suggestions that might improve the paper: In the Methods section the authors mention what volume of PNK enzyme is used for the RNA ligation reactions but they do not specify units. I assume they are using NEB PNK (?) but this comes in various concentrations. I would appreciate it if the authors could also provide the number of units of enzymes in the materials and methods. One potential issue with the method is that it is not possible (or not easy) to determine whether all 5’ ends of the cross-linked RNAs have actually been ligated to their adapter. I agree with the authors that the sequence analysis strongly indicate that the ligation reaction went to completion. Have the authors done any optimization steps for the ligation reaction? It might be worth mentioning that this step may need to be optimized for each protein as the amount of RNA cross-linked to a protein can vary quite significantly. T4 PNK ligase also introduced sequence representation biases as the enzyme seems to have a preference for certain nucleotide combinations. Did the authors have test how well their adaptor ligates to RNAs with different nucleotides at 5’ ends. Most groups now routinely add random nucleotides to adapter sequences to minimize such bias (you can never completely remove it). Have the authors considered doing this? Perhaps the authors could comment on this in the manuscript. The authors have specifically looked at the 5’ ends of the read-through fragments for motif analysis. These results suggest that most of the RNA sequences in read-through fragments have A’s at the 5’ end, which is to be expected as the RNAs were fragmented with RNAse I. I am wondering though if it would not be also interesting to look at mutations within these read-through fragments to assess whether (a) all read-through fragments have mutations, (b) whether these primarily are deletions or substitutions and whether they cluster together. I would also be interested to see if the motifs found at or near deletions/substitutions are similar to the ones identified in the truncated cDNAs. Essentially, these analyses could answer a long-standing question about how reliably mutations are for pin-pointing the UV cross-linking site. The reason for asking this is that we sometimes observe deletions in U-rich regions and it is not always evident whether these deletions are the result of over cross-linking the cells (RNA damage) or whether these are actual UV-cross-linking sites. Because the read-through fragments may have multiple mutations, they may not map accurately to the reference sequence. Can the authors comment on how many of the read-through reads did not or poorly align to the reference sequence?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank the reviewer for the excellent suggestions, and we will provide a complete response along with the revised manuscript once we receive further reviews. For now, I would just like to clarify that this method doesn't work under the assumption that 5’ ends have been efficiently ligated to their adapter. In fact, more likely the opposite is true, since the 5' ends of CLIP RNAs can often be buried in the protein binding pocket, and can form RNA structures that make them inaccessible to PNK phosphorylation and ligation. Thus, we assume that the 5' ligation efficiency is probably below 50%. It would be difficult to increase it much further due to the variable accessibility of RNA fragments.Importantly, high ligation efficiency is not needed for the analyses used in the manuscript, since we focus primarily on those reads that have the adapter - and here we can be confident that the ligation worked, so these certainly are readthrough cDNAs. The purpose of our method is to identify the sequence characteristics of the readthrough RNAs within a standard iCLIP experiment, which maximises the recovery of RNA fragments by amplyfying them regardless of whether 5' marker has been ligated or not.A more thorough response will follow later.