The purpose of this study was to explore the characteristics of pupil offset in young Asian adults with myopia.In total, 1200 eyes  were divided into mild-moderate and high groups according to equivalent spherical diopters (SEQ). The pupil offset and its X and Y components were compared between the groups. Linear correlation was analyzed among pupil offset, X and Y components, and SEQ. Multiple linear regression analysis was conducted for pupil offset and eye parameters.P < 0.05). The magnitude of pupil offset, X and Y components, and SEQ were positively correlated. The pupil center (PC) of the right eye in the mild-moderate group was mainly superotemporal to the corneal vertex and mainly superonasal for the left eye and both eyes in the high group. Multiple linear regression analysis revealed that the magnitude of pupil offset correlated with central corneal thickness, intraocular pressure, and mean corneal curvature (P < 0.05).The mean age of all subjects was 22.5 ± 4.8 years. The mean magnitude of the pupil offset (0.18 ± 0.09 mm vs. 0.15 ± 0.08 mm) and Y component (0.12 ± 0.08 mm vs. 0.10 ± 0.07 mm) were larger in the high group than in the mild-moderate group (The magnitude of the pupil offset that correlated with partial eye parameters and its X and Y components increased as the SEQ increased, and the PC gradually shifted toward the superonasal direction in young Asian adults with myopia.Subjects with high myopia with a larger pupil offset should be considered for better postoperative visual quality during refractive surgery. Although the pupil offset or angle kappa has been well studied in previous studies, different degrees of myopia have not been fully addressed, nor have they completely focused on young adults who are the primary group for refractive surgery. In contrast, this study concentrates on young adults with myopia, which can provide a supplementary study for the above clinical problems. It is clinically important to clarify the characteristics of the pupil offset in myopia. More importantly, it will provide a vital pre-operative reference for surgeons and raise attention to pupil offset in patients with different degrees of myopia. It further provides a suitable surgical design for refractive surgery in young adults and avoids postoperative decreases in visual quality caused by decentered ablation.A total of 600 young patients with myopia  who underwent comprehensive pre-operative examination for refractive surgery were recruited for this cross-sectional study from January 2021 to August 2021 at Tianjin Medical University Eye Hospital.According to the refractive surgery consensus, the inclusion criteria were as follows: (1) the diopter was stable for at least 2 years. (2) Soft contact lenses, hard contact lenses, and orthokeratology were discontinued at least 2 weeks, 1 month, and 3 months before the operation, respectively. (3) The best-corrected visual acuity (BCVA) was >20/25. The exclusion criteria were as follows: active inflammation and serious accessory organ lesions of the eyes, glaucoma, keratoconus, diabetes, serious mental illness, and autoimmune illness.All recruited subjects appreciated the purpose and significance of this study, and written informed consent was obtained from them. The study was approved by the Ethics Committee of Tianjin Medical University Eye Hospital and conformed to the principles of the Declaration of Helsinki (2020KYL-33).According to SEQ (range –0.75 D to –10.00 D), the subjects were divided into the mild-moderate myopia group (–0.75 D ≤ SEQ ≤ –6.00 D) and the high myopia group (–6.25 D ≤ SEQ ≤ –10.00 D). Comprehensive pre-operative ophthalmic examinations were performed for all subjects, including uncorrected visual acuity (UCVA), BCVA, dominant eye, intraocular pressure , slit-lamp examination, dilated fundus examination, apparent optometry, corneal topography, pupil offset, pupil diameter, corneal curvature, and corneal thickness. All examinations and data collection were performed by skilled ophthalmologists and operators under the same conditions.,As stated by Gharaee,Before the measurements, the head positions of the participants were suitably adjusted. During the measurement, the subjects were strongly urged to keep their eyes open, look at the target of the device, and not blink. After the measurement, all subjects were required to close their eyes for a few moments to ensure an even distribution of the tear film for the next examination. Data were accepted only when the quality specification showed OK; otherwise, it was remeasured. The pupil offset was ideally measured three times per eye with eligible data, and the mean value of the three measurements was taken as the final pupil offset of each patient to ensure the reliability of the data.P < 0.05 were considered statistically significant.IBM SPSS Statistics 20  and Prism 9.0 (GraphPad Software) were used for data processing and statistical analysis. The Kolmogorov–Smirnov test was used to test the normality of the data, which are presented as mean ± standard deviation (SD). The UCAV, SEQ, pupil offset, and its X and Y components were compared between the two groups using the generalized estimation equation (GEE), which could be used to analyze nonindependent data measured from both eyes of each patient. Scatter plots (CV as the zero point) with a confidence ellipse were used to describe the distribution of pupil offset in 1200 eyes and to determine the orientation with the greatest SD. A linear correlation analysis was conducted for the pupil offset, X and Y components, and SEQ. In addition, multiple linear regression analyses (stepwise) based on reasonable independent variable screening mechanisms were performed to identify the parameters that were correlated with the magnitude of pupil offset and its X and Y components. Values of P > 0.05, independent-samples t-test; see P > 0.05, chi-square test; see P < 0.01). The mean pupil offset of the high myopia group (0.18 ± 0.09 mm) was larger than the mild-moderate myopia group . Further comparison of the X and Y components was performed via GEE, and the mean value of the Y-component was larger in the high group (0.12 ± 0.08 mm) than in the mild-moderate group (0.10 ± 0.07 mm) with a significant difference (P < 0.05), whereas there was no significant difference in the X-component (P > 0.05).The baseline characteristics and results of 600 subjects with 1200 eyes are shown in P < 0.001). Similarly, as the SEQ increased, the Y-component (P < 0.001) compared with the X-component , the Y-component had a stronger correlation with the SEQ (R2 = 0.014).As shown in omponent C of the As shown in P < 0.05; P < 0.05). Multicollinearity was weak between the parameters, based on the values of variance inflation factor (VIF) around 1.000.The results of the multiple linear regression analysis (stepwise) are summarized in ,The human eye is a complex optical system with various essential optical parameters, such as pupil offset, visual axis, pupillary axis, and angle kappa. Pupil offset, considered to be the decentration between PC and CV, can lead to decentered ablation in refractive surgery.,Pentacam is used to measure the pupil offset and X and Y components in this study as Frings’ procedure, and accurate results of pupil offset can be obtained with this method.The magnitude of pupil offset in the high myopia group was significantly larger than that in the mild-moderate myopia group, which was different from previous studies. For instance, Reinstein et al. found that the largest pupil offset was in the hyperopia group and the smallest was in the myopia group.The pupil offset was linearly and positively correlated with the preoperative SEQ, which was inconsistent with Reinstein et al., who stated that pupil offset in subjects with myopia decreased as the SEQ increased.,The Y-component of pupil offset in the high myopia group was greater than that in the mild-moderate myopia group. The difference in the Y-component between the two groups was similar to the findings of Holden et al.,In the mild-moderate myopia group, the PC was predominantly temporal to the CV in the right eyes and nasal to the CV in the left eyes, which can be explained by the aforementioned right-handedness. In the high myopia group, the PC of both the right and left eyes was predominantly nasal to the CV, which was similar to a study that found that the PC gradually shifted to the nasal side of the CV as the axial length increased.In this study, 40.67% (488) of eyes had a PC that deviated toward the temporal side of the CV, 56.92% (683) of eyes had a PC that deviated toward the nasal side, and 43% (516) of eyes had a superonasal tendency, consistent with Chung et al.,,Multiple linear regression analysis suggested that the pupil offset correlated with CCT, IOP, and mean corneal curvature. The negative correlation between the pupil offset and IOP may be attributed to the fact that increased IOP can lead to changes in pupil and corneal surface morphology, which in turn decreases pupil offset. There was no correlation between pupil offset and age, which differed from the study of Hashemi et al., which may be due to the age range of the patients in this study.A limitation of this study is that pupil offset, CV, and PC were tested using devices based on a two-dimensional screen; however, refractive surgery is a three-dimensional procedure. Further studies on the three-dimensional structure of the pupil offset should be conducted. In addition, only partial ocular anterior segment parameters that might be associated with pupil offset were included in the multiple linear regression analysis, and additional parameters should be investigated in further studies.In conclusion, this study found that pupil offset in myopic Asian eyes of young adults increased with increasing pre-operative SEQ, indicating that pupil offset was greater in subjects with high myopia than in those with mild-moderate myopia. In addition, as the SEQ increased, the PC gradually shifted toward the nasal side of the CV. To improve postoperative visual quality, refractive surgery must be well designed and corrected before and during the surgery due to the presence of pupil offset, especially in young adults with high myopia.


Small RNAs (sRNAs) are an important group of non-coding RNAs that have great potential as diagnostic and prognostic biomarkers for treatment of a wide variety of diseases. The portability and affordability of nanopore sequencing technology makes it ideal for point of care and low resource settings. Currently sRNAs can’t be reliably sequenced on the nanopore platform due to the short size of sRNAs and high error rate of the nanopore sequencer. Here, we developed a highly efficient nanopore-based sequencing strategy for sRNAs (SR-Cat-Seq) in which sRNAs are ligated to an adapter, circularized, and undergo rolling circle reverse transcription to generate concatemeric cDNA. After sequencing, the resulting tandem repeat sequences within the individual cDNA can be aligned to generate highly accurate consensus sequences. We compared our sequencing strategy with other sRNA sequencing methods on a short-read sequencing platform and demonstrated that SR-Cat-Seq can obtain low bias and highly accurate sRNA transcriptomes. Therefore, our method could enable nanopore sequencing for sRNA-based diagnostics and other applications. Small RNAs (sRNAs) are a diverse class of non-coding RNA that include microRNA (miRNA), small nucleolar RNA (snoRNA), tRNA fragments (tRNAf) and piwi-interacting RNA (piRNA) among others. They are important in a variety of cellular functions, including development and regulation of gene expression . FurtherNanopore sequencers, in particular, the commercially available sequencers from Oxford Nanopore Technologies (ONT), offer many advantages for diagnostic applications, including low costs for equipment and consumables. Unlike other sequencing technologies, where data are analyzed after collection, nanopore devices are capable of real time analysis and on-chip target enrichment , making Previous approaches to detect sRNAs on ONT devices involve using chemical modifications to add bulky residues to slow the voltage driven translocation of molecules through the pore and increase the signal, however it is not possible to achieve whole transcriptome sequencing using this method . In anotAll previous approaches using rolling circle amplification to aide in nanopore sequencing have used phi29 DNA polymerase due to its high processivity and strong strand displacement. We reasoned that a highly strand displacing reverse transcriptase (RT) could achieve the same goal directly on RNA templates and would therefore allow for direct amplification of sRNA. One candidate group of enzymes to perform such a function are Group II intron reverse transcriptases. Group II introns are retrotransposons that encode a self-splicing ribozyme as well as a reverse transcriptase. To complete the retrotransposition cycle, the intron-encoded reverse transcriptase must accurately copy its cognate ribozyme which is large and highly structured. While homologous to the retroviral encoded reverse transcriptases typically used in biotechnology, this selection pressure has led to the evolution of high processivity, high fidelity and strong strand displacement activity in the Group II intron RTs .In this paper we show that it is possible to perform rolling circle reverse transcription using Group II intron RTs, with much better performance than retroviral RTs. We developed an efficient sequencing strategy that allows for adapter ligation, circularization and rolling circle reverse transcription of cellular sRNAs without intermediate purifications. The resulting cDNA products can be used directly or further amplified before being sequenced on the ONT MinION device. Finally, we show that our method for converting sRNAs into concatemeric cDNAs (SR-Cat-Seq) can be used to reconstruct high-fidelity and low-bias sRNA transcriptomes from error-prone nanopore data.Circular RNA was prepared using 10 replicates of 20 μl reactions each containing 50 pmol of a 42nt RNA oligo , oligo 1The following reverse transcriptases (RTs) were tested: Avian Myeloblastosis Virus RT , Moloney Murine Leukemia Virus RT , ProtoScript II RT , SuperScript IV RT , WarmStart RTx , Thermostable Group II Intron RT  and Induro RT (NEB M0681). Each reverse transcription reaction was performed under the manufacture’s recommended buffer conditions with 1 μl of the stock concentration of the enzyme. Each reaction contained 1 pmol of either circular or linear RNA oligo , oligo 1The top RNA strand of the adapter , oligo 3For RNA oligos or the miRXplore synthetic reference miRNAs, 5 pmol of RNA was used as input. For human brain RNA, sRNAs (< 200 base pairs) were isolated from 10 μg of total brain RNA using RNA XP beads (Beckman Coulter) following the manufacturer’s protocol. 50 ng of isolated sRNA was used as input. For the SR-Cat-seq workflow, the RNA was first denatured by heating to 70°C for 2 minutes and placed on ice immediately. The ligation mixture was then added to the RNA. The ligation mixture contained 1x T4 RNA ligase buffer (NEB M0204), 20% PEG-8000, 0.05% Tween-20, 20 pmol of the annealed splintadapter (see above) and 200 units of T4 RNA Ligase 2 Truncated K/Q (NEB M0373) in a total volume of 20 μl. The ligation reaction was incubated at 25°C for 1 hour. Subsequently, 2 units of USER enzyme (NEB M5505) and 4 units of DNase I (NEB M0303) were added to the reaction mixture and incubated at 37°C for 30 minutes to remove the bottom DNA splint strand of the adapter. After the degradation of DNA strand, only RNA is subject to the following circularization step. The T4 RNA Ligase 2 Truncated K/Q was then heat inactivated at 75°C for 5 minutes and then cooled down to 4°C. The reaction mixture was then diluted to a total volume of 40 μL with 1x T4 RNA ligase buffer, 1 mM ATP, 10 units of T4 Polynucleotide Kinase (NEB M0201) and 30 units of T4 RNA ligase 1 (NEB M0437M). The circularization reaction was then allowed to proceed for 1 hr at 25°C. Linear RNAs were then degraded by adding the following mixture of enzymes 2uL XRN-1 (NEB M0338), 1uL 5’deadenylase (NEB M0331), RNase R (purified in house), ATP and Poly(A) Polymerase (NEB M0276L). The reaction mixture was then diluted to 80 μL with 2x Induro RT buffer , 1 μM primer , oligo 2Second strand synthesis was performed using Taq DNA polymerase. A primer , oligo 5Amplification was performed using multiple strand displacement amplification with forward and reverse primers designed to anneal to non-overlapping portions of the adapter sequence (https://www.ncbi.nlm.nih.gov/sra) under project accession number: PRJNA603337. qPCR data was previously published and available in supplementary materials [Short read sequencing data were previously published  and avaiaterials .Adapter ligation and circularization reactions were carried out as described above using a 21 bp randomized RNA substrate with an internal FAM label , oligo 8https://www.R-project.org/) were used to parse the resulting genbank files from the SPADE output to collect the consensus sequences. Consensus sequences generated in this manner could have any random circular orientation, therefore, to phase them we generated all possible rotations of each consensus sequence and aligned the adapter to them using pairwise alignments with the Needleman-Wunsch algorithm as implemented in the R package Biostrings  We chose the first rotation of the sequence that gave the longest un-gapped alignment anchored to either the start or end of the read. The adapter sequence was then trimmed from the rotated consensus sequences to yield the final trimmed consensus sequences.Reads were filtered by length (> 1000 bp) and average quality (> = 7) and then converted to FASTA format. SPADE  was usedhttps://bioconductor.org/packages/Biostrings.) We then determined the edit distance between the consensus sequence and the reference including gaps, insertions, and mismatches. The edit distance was subtracted from the number of aligned bases to determine the percent identity to the reference sequence.We assessed consensus sequence accuracy using a synthetic 21 nt oligo with the sequence of human miRNA hsa-let7a , oligo 1MiRXplore libraries were mapped to the miRXplore reference using bbmap and read counts were generated during mapping. Human RNA libraries were mapped to the human genome (GRCh38) using bowtie  with a sConsidering the 962 miRNAs in miRXplore were present in equimolar amounts, an expected read count was generated by dividing the total number of mapped reads for each library by 962. Reads were then normalized by dividing the raw read counts by the expected read counts. An miRNA represented with the exact expected read count would have a normalized value of 1; over- and under-represented sequences would have values greater and lower than 1, respectively.in silico designed RT with RNase H activity). Two other tested RTs were derived from Group II introns, including thermostable Group II intron RT (TGIRT) and Induro RT. We performed reverse transcription with each RT under the manufacture’s recommended reaction conditions with either a linear or a circularized RNA template. Both linear and circular RNA templates are 42 nt long and share the same sequence. Reaction products were treated with RNase H and RNase If to remove the RNA template and visualized using both agarose gel electrophoresis (for large cDNA products) and denaturing polyacrylamide gel electrophoresis (for short cDNA products). Retroviral RT reactions were allowed to continue for one hour while Group II intron RT reactions were stopped at 15 minutes, because pilot experiments showed that a full hour incubation with the Group II intron RTs would create cDNA products too large to be resolved by the gels. When comparing different RT products resolved by agarose gel electrophoresis, we found that the Group II intron RTs generated larger cDNA products with the circular RNA template, indicating robust rolling circle reverse transcription , Moloney Murine Leukemia Virus RT (MMLV), ProtoScript II RT , SuperScript IV RT  and WarmStart RTx . Subsequently, the DNA splint strand of the adapter is enzymatically degraded. The ligated small RNAs are then diluted to favor intramolecular ligation and circularized with the addition of T4 RNA ligase, followed by a cocktail of exoribonucleases to degrade any remaining linear RNA. The reactions were then diluted a second time to accommodate the buffer requirement for the next step of rolling circle reverse transcription, which was performed in the same tube with addition of Induro RT . The resTo assess the efficiency of RNA circularization in the SR-Cat-Seq workflow, we used a 21-mer, FAM labeled, degenerate RNA oligo , oligo 8To assess the sequencing accuracy of the SR-Cat-Seq workflow, we sequenced a 42-mer RNA oligo, containing the sequence of a human miRNA (hsa-let7a) on the 5’ end and our adapter sequence on the 3’ end, mimicking the ligation product in the first step of the workflow with a fully known sequence. This oligo was circularized and rolling circle reverse transcription was performed using our optimized workflow. The resulting concatemeric cDNA underwent either second strand synthesis (SR-Cat) or amplification by phi29 DNA polymerase (SR-Cat-Amp). We sequenced the libraries and applied the SPADE algorithm  to identTo evaluate the sequencing bias of the SR-Cat-Seq workflow, we sequenced a control set of RNA oligos called miRXplore. MiRXplore is a pool of 962 synthetic miRNAs which are mixed at equimolar concentration and commonly used to benchmark bias in small RNA sequencing workflows. To quantify the sequencing bias, we normalized the read counts such that reads represented in the library at their expected values would have a normalized count of 1, while over-represented reads had a normalized count > 1 and under-represented reads had a normalized count < 1 . The biaWe then applied the workflow to sequence small RNA in human brain samples. The same brain RNA was sequenced with 2 technical replicates for both the SR-Cat and SR-Cat-Amp workflows. We detected various sRNAs such as miRNA, piRNA and tRNA as well as some long non-coding RNA and ribosomal RNA . Both SRIn this work we show that Group II intron RTs have strong strand displacement activity and are capable of quickly producing large concatemeric cDNAs from circular RNA templates. Based on this activity we developed SR-Cat-Seq, a highly efficient one-pot workflow that enables sRNA sequencing on the nanopore platform. The whole workflow includes adding adapters to sRNAs, circularization and rolling circle reverse transcription to produce concatemeric cDNAs from small RNAs. We showed that SR-Cat (without amplification) workflow has minimal sequencing bias, which is comparable to the Low-Bias workflow. However, the amplification step in the SR-Cat-Amp introduced bias to the whole workflow, which remains to be improved. Workflows with low sequencing bias are important in applications such as liquid biopsy where accurate abundance measurements can help to rank targets and guide real-time treatment decisions. Finally, we applied the workflow to sequence small RNAs from human brain RNA and demonstrated similar accuracy and sensitivity to Illumina’s TruSeq, the standard short-read method. In addition, we developed a bioinformatics workflow that can reconstruct highly accurate consensus sequences from the concatemeric cDNA products. High fidelity sequencing is extremely important for small RNA research. Because of the small size of small RNAs, even single mismatch may make mapping difficult and cause incorrect counts. Furthermore, for liquid biopsy applications, tumor specific isomirs are of great interest and high-fidelity sequencing is required for detection.We tested versions of our workflow with and without amplification and found that there were advantages and disadvantages to each. The unamplified workflow (SR-Cat) had the lowest bias and would therefore be the best option to use if an accurate representation of the sRNA transcriptome was desired. However, it also had disadvantages including high input requirements of at least 5 pmol for oligonucleotides or 50 ng of sRNA purified from total RNA. These input requirements would preclude the use of this method for most patient samples without further development. Furthermore, the unamplified workflow had a much higher fouling rate of the nanopores which limited the overall output from each flowcell . While sin situ sequencing [The ability of Group II Intron RTs to perform rolling circle amplification has many possible applications beyond the one explored in this work. Analogous activity in DNA polymerases, such as phi29 DNA polymerase, has enabled many different technologies such as whole genome amplification [quencing  among maS1 Fig(TIF)Click here for additional data file.S2 FigCumulative number of reads sequenced is shown on the y-axis vs time in hours on the x-axis, each line represents an individual MinION flow cell.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


This paper describes a laboratory protocol to perform the NanoString nCounter Gene Expression Assay from nasopharyngeal swab samples.  It is urgently necessary to identify factors related to severe symptoms of respiratory infectious diseases, such as COVID-19, in order to assess the possibility of establishing preventive or preliminary therapeutic measures and to plan the services to be provided on hospital admission. At present, the samples recommended for microbiological diagnosis are those taken from the upper and/or the lower respiratory tract.  The NanoString nCounter Gene Expression Assay is a method based on the direct digital detection of mRNA molecules by means of target-specific, colour-coded probe pairs, without the need for mRNA conversion to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. This platform includes advanced analysis tools that reduce the need for bioinformatics support and also offers reliable sensitivity, reproducibility, technical robustness and utility for clinical application, even in RNA samples of low RNA quality or concentration, such as paraffin-embedded samples. Although the protocols for the analysis of blood or formalin-fixed paraffin-embedded samples are provided by the manufacturer, no corresponding protocol for the analysis of nasopharyngeal swab samples has yet been established. Therefore, the approach we describe could be adopted to determine the expression of target genes in samples obtained from nasopharyngeal swabs using the nCOUNTER technology. The samples currently recommended for the microbiological diagnosis of respiratory infectious diseases are those obtained from the upper respiratory tract (nasopharynx and oropharynx) and/or the lower respiratory tract, such as sputum, endotracheal aspirates, bronchoalveolar lavages or bronchial aspirates, especially in patients with severe respiratory disease.Nasopharyngeal swabs provide a valuable mixture containing biological material both from the infectious agent and from the patient. The high viral load obtained,® platform  delivers direct and multiplexed measurement of gene expression, providing digital readouts of the relative abundance of mRNA transcripts simultaneouslyIn recent years, the study of selected genes by real-time PCR or genome-wide gene expression microarray analysis has been used in genetic research to detect associations between specific gene expression profiles and particular diseases. Within these technologies, the nCOUNTERThe nCOUNTER Human Immunology V2 CSO panel, which facilitates the study of 594 genes, including the major classes of cytokines, chemokine ligands, interferons and their receptors, the TNF-receptor superfamily, the KIR family genes and genes involved with the anti-fungal immune response, is recommended for the study of the immune response to infectious disease in samples with fragmented RNA or low RNA inputs.Our study included 250 patients admitted to the Hospital Costa del Sol  with severe COVID-19 and positive PCR results for SARS-CoV-2. To participate in the study, all patients received a patient information sheet and were asked to sign the corresponding informed consent form.The procedure for the routine determination of SARS-CoV-2 by PCR includes taking a nasopharyngeal sample with the sterile, fine, flexible swab that is included in the specific respiratory sampling kit for viruses. According to the protocol stipulated by the Spanish Ministry of Health,Various kits are currently marketed for the collection, transport, and maintenance of clinical samples until the laboratory analysis is performed, some of which include transport media with an inactivator. In the subsequent analysis of the results, we also considered whether the use of one or other viral transport mediums affected the final result.To safely handle biological samples, they must first be inactivated. With samples obtained from nasopharyngeal swabs for molecular analysis, this is usually accomplished by the addition of a chemical quencher or by heat treatment.,Given the low concentration of genetic material in nasopharyngeal swabs, together with the high presence of biologic contaminants in upper respiratory airways, we recommend heat-treatment inactivation. Various techniques have been described to perform this task without affecting the integrity of the RNA, including inactivation at 56°C for 30 minutes, at 65°C for 15 minutes, at 95°C for 5 minutes or at 98°C for 2 minutes.Before processing the samples, we ensured that thermal inactivation did not impair RNA integrity, by comparing the performance of RT-PCR analysis for SARS-CoV-2 after treating a set of samples to each of the heat inactivation protocols. In our experiment, all samples were inactivated at 98°C for 2 minutes as previously described.Following viral inactivation, RNA was immediately extracted. Given the novelty of the protocol, we performed a local validation of a subset of samples to assess the performance of both the automated and manual RNA extraction procedures.For automated extraction, we tested the performance of the Biorobot EZ1 , with an initial sample volume of 200 μl and a final eluate volume of 60 μl; and the MagCore robot (Magcore Lamination India PvT. Ltd), with initial sample volumes of 200 μl or 400 μl and a final eluate volume of 40 μl. Although these kits are not the most optimal methods to allow for the detection of host genes, our preliminary results demonstrate their validity to study host gene expression.With respect to the manual RNA extraction procedure, and due to supply shortages during the COVID-19 pandemic, we were not able to use the recommended QIAMp MinElute Virus extraction kit (Qiagen). Instead, and according to previously published papers in this field, we tested the RNEasy Mini Kit (Qiagen), which is one of the most common commercial kits and the Gold Standard for RNA extraction, as an alternative strategy for viral detection in sputumTM2000 spectrophotometer . NanoString input recommendations stipulate a total RNA concentration range of 100-130 ng and specific ratios of absorbance at 260 nm and 280 nm (A260/280) measured with NanoDrop within the range 1.80-2 nm. Since aromatic proteins have a strong UV absorbance at 280 nm, the A260/280 ratio is generally used to assess protein contamination in a nucleic acid sample. A260/280 ratios under 1.7 usually indicate the presence of contaminants that can affect the result, being the A260/280 ratio of ~2.0 as the generally accepted as “pure” for RNA. In such a manner, the A260/230 ratio is normally used to reveal the presence of organic contaminants such as phenol or TRIzol. Generally acceptable A260/230 ratios are those in the range of 1.8-2.2.Given its affordability, rapidity an ease of use, we firstly carried out a general assessment of eluates sample quality and concentration with the NanoDropIn our local validation study, we obtained varying results for RNA concentration and absorbance ratios  for eachTM2000 spectrophotometer as well as RNA concentration and integrity measured with the Agilent 2100 System Bioanalyzer  of a subset of 131 purified RNA aliquots from heat-inactivated nasopharyngeal swabs (2 minutes at 98°C) processed with the MagCore robot , considering the different viral transport media brands in which patient samples were preserved   .After heat inactivation and RNA extraction, RNA eluates were stored at -80°C until further processing. Samples were prepared following manufacturer’s instructions and diluted, in the case of the most concentrated samples, so that the final volume of 5 μl per well contained 150-200 ng of total RNA. Samples were hybridized 22 hours at +65°C and stored at +4°C until digital readout. Results were obtained with the nCounter Prep Station and Digital Analyzer set at high sensitivity. Performance of the NanoString platform and panel validity was assessed with respect to positive and negative controls as well as to the number of readings of housekeeping genes .The differential expression analysis data model preferentially applies the optimal statistical method per gene given the following variable distribution: 1) Mixture negative binomial model, 2) Simplified negative binomial model, 3) Log-linear model, in that order. FDR p-value adjustment will be performed according to the Benjamini-Hochberg method. All results are normalized using the geometric mean of the housekeeping genes.Our study had several strengths, as being the first study in to set up a protocol for the digital multiplexed gene expression analysis of nasopharyngeal exudates using the NanoString nCounter System. On the other hand, and apart from the lack of previous studies on this topic, our study also had other limitations: Since our study is based on the analysis of diagnostic surpluses, we have only been able to analyze a certain number of samples preserved in each viral transport medium. We neither know which of the original nasopharyngeal samples were stored at -80°C and subsequently thawed before performing the diagnostic PCR during the care process, which could explain why RNA in some samples was quite degraded. Finally, reagents shortage during the first and second waves of the COVID-19 pandemic, has also conditioned the techniques used.COVID-19 is a major global health problem, making it necessary to develop tools to optimize healthcare and facilitate personalized treatment. From a clinical perspective, the identification of gene transcripts related to the poor prognosis of patients hospitalized with SARS-CoV-2 has undeniable practical value. This complementary information would be straightforward to design multiplexed panels and prediction tools that can be incorporated into computers used in daily practice, helping clinicians predict and identify possible outcomes and facilitating decision-making in this respect.Our study may also provide useful information to help establish the protocols of other studies based on RNA analysis from nasopharyngeal swab samples using the NanoString nCOUNTER platform.Nanostring nCounter nSolver™ 4.0 (RRID:SCR_003420), using the Nanostring Advanced Analysis Module 2.0 plugin and following the Nanostring Gene Expression Data Analysis Guidelines. Advanced Analysis Module 2.0 pluginhttps://www.nanostring.com/products/analysis-solutions/ncounter-advanced-analysis-software/ and following the NanoString Gene Expression Data Analysis Guideliness. Advanced Analysis Module 2.0 software uses open-source R programs for quality control, normalization, differential data analysis, pathway scoring and gene-set enrichment analysis.Normalization, differential expression analysis and pathway analysis can be performed withMGA, SGR, IBM, MR conceptualization of the study. SGR, MGA, MR funding acquisition. MGA, ILR, TD, VDL, IBM, MA, MLH investigation and methodology. MPR, IBM, MGA, MA contributed to data analysis. MGA, IBM, MR supervised the study and revised the manuscript. All authors read and approved the final manuscript.Our Institutional Review Board (CEI Costa del Sol exp.003_JUL20_PI-IMMU-COVID19) approved this study in July 2020. All patients were informed of the study and invited to participate. All participation was subject to the provision of informed written consent. The authors have adequately responded to my comments/concerns. I do not have further comments.Is the rationale for developing the new method (or application) clearly explained?YesIs the description of the method technically sound?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?PartlyIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Organ transplantation, kidney, Biomarker, Transcriptomics, ProteomicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Authors have satisfactorily addressed most of my concerns and explained why they used less than optimal approach. They included a paragraph with limitations that explains the large variations in RNA quantity and quality. It is clear that more samples and prepared in more controlled approach are necessary before we can conclude that nasopharyngeal swabs can be used for NanoString nCounter Gene Expression Assay.In Table 2 Copan UTM-RT row, put correct SD for A260/280 and A260/280 ratios. Minor issues:Is the rationale for developing the new method (or application) clearly explained?YesIs the description of the method technically sound?PartlyAre the conclusions about the method and its performance adequately supported by the findings presented in the article?NoIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?PartlyAre sufficient details provided to allow replication of the method development and its use by others?PartlyReviewer Expertise:Virology and molecular biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The method on the digital multiplexed gene expression analysis of nasopharyngeal swab samples using the NanoString nCounter system is adequately presented in the manuscript. This could be of interest to the readers of F1000Research and researchers working with gene expression of immune signature and SARS-CoV-2. The authors should provide information on any failure in extracting enough total RNA as demanded by the method.In real-life situations it is hard to get enough RNA from 100% samples. If it was the case then it has to be stated clearly.There is some ambiguity on the amount of total RNA. From the manuscript it appears that only 100 ng is needed. The following statement is contradicting: "4. Add 5 μl of the samples to each corresponding well (RNA concentration should be 100 μg/μl. Dilute out-of-range samples with RNAse-free water)."  I find the rationale of the protocol adequate. I find the protocol not quite complete. Such as: Is the rationale for developing the new method (or application) clearly explained?YesIs the description of the method technically sound?YesAre the conclusions about the method and its performance adequately supported by the findings presented in the article?PartlyIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?YesAre sufficient details provided to allow replication of the method development and its use by others?YesReviewer Expertise:Organ transplantation, kidneyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We thank the reviewer for the constructive comments which will help us to improve the quality of our work. We hope that the revisions in the manuscript and our accompanying responses will be sufficient to make our manuscript suitable for acceptance. Please find below our point-to-point responses to the comments. The method on the digital multiplexed gene expression analysis of nasopharyngeal swab samples using the NanoString nCounter system is adequately presented in the manuscript. This could be of interest to the readers of F1000Research and researchers working with gene expression of immune signature and SARS-CoV-2. The authors should provide information on any failure in extracting enough total RNA as demanded by the method. In real-life situations it is hard to get enough RNA from 100% samples. If it was the case then it has to be stated clearly.We agree with the Reviewer and will include the success rate of the extractions performed with the MagCore robot: The success rate of extractions performed with the MagCore robot has been 86%.There is some ambiguity on the amount of total RNA. From the manuscript it appears that only 100 ng is needed. The following statement is contradicting:  "4. Add 5 μl of the samples to each corresponding well (RNA concentration should be 100 μg/μl. Dilute out-of-range samples with RNAse-free water)." We agree with the reviewer and apologize for error. We will modify the corresponding paragraphs and will also include additional information:NanoString input recommendations stipulate a total RNA amount range of 100-300 ng and specific ratios of absorbance at 260nm and 280nm (A260/280) measured with NanoDrop within the range 1.8-2nm. Given the characteristics of the samples, we decided to hybridize the nCounter probes with 100 ng to 200 ng of total RNA per assay, reason why we only assessed gene expression on eluates with total RNA concentration > 2.5 ng/µl measured with the bioanalyzer.Samples were prepared following manufacturer’s instructions. When necessary, we diluted samples in order to obtain 100-200ng of total RNA to each corresponding well. Time of hybridization was set between 16 and 21 hours. Results were obtained with the nCounter Prep Station and Digital Analyzer set at high sensitivity. I find the rationale of the protocol adequate. I find the protocol not quite complete. Such as:  Authors here described their protocol for RNA preparation from nasopharyngeal swabs for subsequent analysis using the NanoString nCounter Gene Expression Assay. This article is a combination of method development with a description of a study on 250 SARS-CoV-2 positive patients with severe COVID-19.Major issuesThe major criticism here is that readers will not be able to assess if the RNA preparation from nasopharyngeal swab and subsequent evaluation by NanoString platform delivered good and reliable data. Reviewer assumes that it will be part of future publication, but without this data it is difficult to judge that nasopharyngeal swabs can be used as source material for this platform. Ideally, authors can post a preprint into archives and put link here.Authors compared manual RNEasy kit that purifies total RNA  with automated kits specialized for purification of viral nucleic acid. Authors should have used e.g. QIAamp MinElute Virus kit or similar for a fair comparison.Authors mentioned the nCounter Human immunology V2 CSO panel that can analyze 594 host genes. For this panel, total RNA isolation kit would be a better choice.Did authors test the platform also with the RNAs purified from EZ1 Virus Mini kit? The A260/A230 ratio is not very good, but high A260/A280 does not necessarily indicate contaminants. Did authors get chance to check the whole UV range spectrum?It would be beneficial to show more data about different viral transport medium, heat inactivation, different nucleic acid extraction protocol and compare their impact on results from several internal controls, rather than showing manufacturer’s instruction for NanoString assay. Was there really any change in the protocol in the case of RNA from nasopharyngeal swabs? If yes, please stress the differences only.Minor issues:Authors should change the future tense to past tense when describing their study in paragraphs “Patients”, “Data analysis” and “Ethics”.Table 1 correct ARN to RNA.Include number of samples “n” per each isolation method in the Table 1.Make clear protocol point no. 4. At the protocol beginning, it is mentioned using 100ng of total RNA, but no.4 talks about using 5µl of sample and RNA concentration 100µg/µl.In the protocol include temperature and other details (such as conditions of each spin).Is the rationale for developing the new method (or application) clearly explained?YesIs the description of the method technically sound?PartlyAre the conclusions about the method and its performance adequately supported by the findings presented in the article?NoIf any results are presented, are all the source data underlying the results available to ensure full reproducibility?PartlyAre sufficient details provided to allow replication of the method development and its use by others?PartlyReviewer Expertise:Virology and molecular biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. We thank the reviewer for taking the time and effort necessary to review the manuscript and sincerely appreciate all valuable comments and suggestions which will help us to improve the quality of the paper. Please find below our point-by-point responses to each of the comments: Authors here described their protocol for RNA preparation from nasopharyngeal swabs for subsequent analysis using the NanoString nCounter Gene Expression Assay. This article is a combination of method development with a description of a study on 250 SARS-CoV-2 positive patients with severe COVID-19.Major issuesThe major criticism here is that readers will not be able to assess if the RNA preparation from nasopharyngeal swab and subsequent evaluation by NanoString platform delivered good and reliable data. Reviewer assumes that it will be part of future publication, but without this data it is difficult to judge that nasopharyngeal swabs can be used as source material for this platform. Ideally, authors can post a preprint into archives and put link here.We agree with the reviewer. However, we would like to point out that, as a pre-protocol study, we only have preliminary results. We are currently generating additional data and we will be able to stablish more consolidated conclusions in the protocol paper.Authors compared manual RNEasy kit that purifies total RNA  with automated kits specialized for purification of viral nucleic acid. Authors should have used e.g. QIAamp MinElute Virus kit or similar for a fair comparison.We agree with the reviewer, however, due to shortage of RNA extraction kits during the COVID-19 pandemic, we were not able to use the QIAmp MinElute Virus kit. Instead, we decided to use RNeasy Mini kit based on previously published papers reporting its use as a valid alternative strategy for viral detection in sputum (DOI: 10.1016/s0166-0934(01)00284-1)or nasopharyngeal samples (https://www.biorxiv.org/content/10.1101/2020.03.20.001008v1.full.pdf), Besides, the RNeasy Mini Kit is one of the most common commercial kits and the Gold Standard for RNA extraction. We will modify the corresponding paragraphs in the text accordingly.Authors mentioned the nCounter Human immunology V2 CSO panel that can analyze 594 host genes. For this panel, total RNA isolation kit would be a better choice.We agree with the reviewer. Indeed, we used RNEasy kit that purifies total RNA. We will modify the corresponding paragraph to make this point clearer.Did authors test the platform also with the RNAs purified from EZ1 Virus Mini kit? The A260/A230 ratio is not very good, but high A260/A280 does not necessarily indicate contaminants. Did authors get chance to check the whole UV range spectrum?We are sorry we did not. Despite its affordability, rapidity and ease of use, the NanoDrop usually does not give perfect and reliable readings, especially in samples with contaminants. For this reason, we measured the A260/280 ratios to get a general idea about the best RNA extraction method within the equipment available in the molecular biology laboratory of our hospital. Once we chose the method that best suited our criteria of 260/280 ratios (1.80-2.0 nm), we assessed the quality and quantity of RNA eluates with the Agilent 2100 Bioanalyzer. We will include a new table with the corresponding results in the manuscript.  It would be beneficial to show more data about different viral transport medium, heat inactivation, different nucleic acid extraction protocol and compare their impact on results from several internal controls, rather than showing manufacturer’s instruction for NanoString assay. Was there really any change in the protocol in the case of RNA from nasopharyngeal swabs? If yes, please stress the differences only.Following the recommendations of the Reviewer, we have added information regarding the impact of transport medium and the heat inactivation protocol. While the former did not influence the experiment performance, the heat inactivation protocol was selected based on the reported standards (DOI: 10.1099/jgv.0.001539). Regarding our in house modifications of the manufacturers protocol to adapt it to this specific type of sample, as the reviewer emphasizes, it is important to make them clear in the manuscript. Therefore, we have modified it including and highlighting the optimized processes. This is of high relevance given that RNA from remnant nasopharyngeal exudates is present at very low concentrations and is highly degraded; indeed the RNA integrity number was less than 6 and the DV200 was less than 30% in most of the samples, what would make them inadequate for RNAseq. These samples are also highly heterogeneous, present great variability from patient to patient, and have been transported with several types of transportation medium. Also sampling procedure varies from center to center. Moreover, the fact that only diagnostic remnants were used implied that only limited volumes were available. For these reasons, the optimization of such a method that detects enough number of genes with this type of diagnostic residual samples is of high importance for performing research in the field.Minor issues:Authors should change the future tense to past tense when describing their study in paragraphs “Patients”, “Data analysis” and “Ethics”.The original version of the manuscript corresponds to the development phase of the technique, which was carried out prior to patient recruitment, reason why “Patients” and “Ethics” sections were written in future tense. We will modify the corresponding paragraphs accordingly.Table 1 correct ARN to RNA.We apologize for the mistake and will correct the typo in the new version of the manuscript.Include number of samples “n” per each isolation method in the Table 1.We appreciate the Reviewer’s suggestion, and will include the corresponding information in the new version. Make clear protocol point no. 4. At the protocol beginning, it is mentioned using 100ng of total RNA, but no.4 talks about using 5µl of sample and RNA concentration 100µg/µl.We apologize for the mistake and will change the corresponding paragraph.In the protocol include temperature and other details (such as conditions of each spin).We agree with the Reviewer’s suggestion and will modify the protocol accordingly.


Nidovirales is a diverse group of (+)RNA viruses, with a common genome organization and conserved set of replicative and editing enzymes. In particular, RNA methyltransferases play a central role in mRNA stability and immune escape. However, their presence and distribution in different Nidovirales families is not homogeneous. In Coronaviridae, the best characterized family, two distinct methytransferases perform methylation of the N7-guanine and 2′-OH of the RNA-cap to generate a cap-1 structure (m7GpppNm). The genes of both of these enzymes are located in the ORF1b genomic region. While 2′-O-MTases can be identified for most other families based on conservation of both sequence motifs and genetic loci, identification of the N7-guanine methyltransferase has proved more challenging. Recently, we identified a putative N7-MTase domain in the ORF1a region (N7-MT-1a) of certain members of the large genome Tobaniviridae family. Here, we demonstrate that this domain indeed harbors N7-specific methyltransferase activity. We present its structure as the first N7-specific Rossmann-fold (RF) MTase identified for (+)RNA viruses, making it remarkably different from that of the known Coronaviridae ORF1b N7-MTase gene. We discuss the evolutionary implications of such an appearance in this unexpected location in the genome, which introduces a split-off in the classification of Tobaniviridae.The order  Nidovirales order encompasses a remarkably diverse group of enveloped, positive-sense RNA viruses with genome sizes ranging from ∼11 to 41 kb. The order is currently classified into 14 families, with members infecting a range of hosts (eight vertebrate and six invertebrate families)  under populated families such as the Mononiviridae (>41 kb). Despite these significant size-differences, these viruses are classified together based on their relatively conserved genome organization and mRNA transcriptional scheme. In the majority of nidoviruses, the first two-thirds of the genome contains two large overlapping open reading frames (ORF1a and 1b). Translation of the ORFs yields two polyproteins (pp1a and pp1ab) containing between 12 and 16 non-structural proteins involved in viral replication and transcription. The 3′ end of the genome codes for a nested set of subgenomic mRNAs (sg mRNAs) directing the synthesis of structural and accessory proteins. The name of the order comes from Nido (latin: nest), which refers to this common ‘nested’ gene expression pattern allowing differential expression levels of non-structural versus structural and accessory proteins (The amilies) . At one Nidovirales RNA genomes and transcripts need both genetic and biophysical stability in order to faithfully transmit and express their genetic content. Genetic stability is generally ensured by the replication/transcription complex (RTC), composed of a core RNA-dependent RNA polymerase (nsp12) flanked with processivity factors nsp7 and nsp8  as the methyl donor, converting it to S-adenosyl-homocysteine (AdoHcy or SAH). Methylation generally occurs in a sequential order, with the N7-guanine methylated first for generation of the mGpppN-RNA (type-0 cap), followed by the 2′-hydroxyl position of the first transcribed nucleotide, yielding mGpppNm-RNA (type-1 cap).For the nidoviruses, the presence of an RNA cap has been reported for three distinct viruses within the order , but higMost large RNA viruses >17 kb) of positive genome polarity generally encode at least one MTase gene, and it has been proposed that the correlation between RNA genome size and presence of an MTase reflects a beneficial coevolution  kb of po. Class ICoronaviridae, most of the enzymes required in this capping pathway have now been identified, including the N7-guanine and 2′-O MTases, which reside in nsp14 and nsp16, respectively , and can be further characterized by their RF structure and K–D–K–E catalytic tetrad. Unlike 2′O-MTases, RF N7-MTases lack a specific catalytic signature sequence. As such, determining their methylation-site specificity is more challenging. Furthermore, the N7-MTase domain residing in nsp14 of CoVs shows very limited sequence conservation with other N7-MTases, and lacks the βαβ architecture characteristic of RF enzymes. While nsp14-like, non-RF N7-MTases can also be identified for several other nidoviruses, this enzyme appears to be lacking entirely for many of the families. Interestingly however, we and others have identified a putative N7-MTase in pp1a (N7-MT-1a) of several members of the large-genome Tobaniviridae family  tobanivirus members  was synthesized by GenScript, with EcoRI and HindIII restriction enzyme sequences flanking the 5′ and 3′ ends respectively. The synthesized gene was subcloned into a pQE30 expression vector, under the control of a T5 promoter, incorporating a 5′ hexahistidine sequence. Deletion PCR mutagenesis with outward facing primers was subsequently performed using Q5 site-directed mutagenesis (New England Biolabs), to delete regions from the 5′ end region. Final recombinant plasmids contained domains starting from Met1333, Thr1342, Thr1350, Ser1366, Ala1358, Asp1374 or Ser1389, spanning to Ile1668. A final C-terminal truncation was additionally performed for expression of a 260 amino acid (∼30 kDa) domain spanning from Asp1374–Asp1633. This construct was subsequently used for site-directed mutagenesis using outwards facing, overlapping primers and High Fidelity Phusion Polymerase (NEB).An E. coli overnight at 18°C from cultures induced with 0.4mM isopropyl-β-d-thiogalactopyranoside and 2% ethanol once the OD600 reaction 0.8. Bacterial pellets were resuspended in lysis buffer , incubated for 30 min before lysis via sonication . The soluble lysate was purified with affinity chromatography using HisPur Cobalt resin (Thermo Scientific). The bound lysate was washed with 2 column volumes wash buffer  and eluted with 150 mM imidazole . The purified protein was subsequently concentrated and loaded onto a Superdex 200 gel filtration column , eluted in gel filtration buffer .Protein was expressed in C2523 (pLacI) O-pivaloyloxymethyl phosphoramidite ribonucleotides , the 5′-hydroxyl group was phosphorylated and the resulting H-phosphonate derivative was oxidized and activated into a phosphoroimidazolidate derivative  was used to transfer the reactions to 8 × 12 filtermats  coated with a 0.3% (w/v) solution of polyethylenimine  for RNA binding. Filtermats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol, prior to soaking in liquid scintillation fluid. RNA methylated with 3H-AdoMet was measured using a Wallace MicroBeta TriLux Liquid Scintillation Counter (Perking Elmer).Methylation activity was performed using filter binding assays, with reactions prepared in 96-well plates. For standard assays, final buffer conditions were 40 mM Tris pH 8, 1 mM DTT, 0.7 μM RNA, 1.9 μM cold AdoMet, supplemented with 0.1 μM 12 or pppA-RNA12, 10 μM) were capped using the vaccinia virus capping enzyme (New England Biolabs) in the presence of 2 μCi α-32P-GTP (Perkin Elmer) to yield radiolabeled G*pppN capped RNA (where * represents the radioactive α-32P). Duplicate reactions were run in the presence of 0.1 mM AdoMet, for creation of N7-guanine methylated cap controls (mG*ppppN). Additional reactions were additionally prepared using pppNm-RNA12 (pre-methylated at the 2′O position), in the presence or absence of AdoMet, for generation of G*pppNm and mG*pppNm cap controls. Capped RNA was incubated with 1 μM  of the purified WB MTase enzyme in a final reaction containing 40 mM Tris, pH 8, 40 μM AdoMet, and 1 μM capped RNA. RNA was digested with Nuclease P1 (New England Biolabs) for 3–4 h at 37°C, and loaded onto polyethylenimine cellulose TLC sheets (Macherey Nagel), along with cap controls. Samples were separated using 0.65 M LiCl as mobile phase. TLC sheets were exposed and visualized using using an Amersham Typhoon BiomolecularImager .Triphosphorylated RNA (pppG-RNA1374–Asp1633), mixed with AdoMet (New England Biosciences) at a 1:1.2 ratio prior to crystallization. All crystals were grown at 293.15 K, using a 1:1 ratio of protein (8 mg/mL) to precipitant solution , and grew in around 5 days as long needles/urchins. The presence of isopropanol drastically improved the size and diffraction of crystals. In our attempt to phase, several crystals were soaked in Tantalum Bromide and harvested while green, according to manufacturer protocol (Jena Bioscience). All crystals were cryo- protected with reservoir solution supplemented with 20% PEG400, and flash-frozen in liquid nitrogen at 100 K. Data collection and diffraction data were collected at Soleil synchrotron, from several crystals. Original native and Sulfure SAD data set were collected on Proxima2, under the assumption that we could use experimental phasing to solve the structure. Further Sulfure SAD and Tantalum SAD data were collected on Proxima1. Data sets were processed individually and analysed with autoPROC toolbox . Conditions were optimized for the WBV 260 construct , human (PDB 3BGV) and Vaccinia virus (PDB 2VDW) as the closest homologues . Structural superimpositions of these structures was done manually using UCSF CHIMERA . The MSA was then manually modified (respecting amino acid chemistry and with no random gap introduction) based on the most conserved motifs/residues, determined from the extended structural MSA profile. Secondary structure prediction using PHD  , Asp1374 (WBV-295) or Ser1389 (WBV-280) produces similar yields of soluble proteins. However, all four purified expression products appear as a double-band at their approximate molecular weights (31–34 kDa) when analyzed by SDS-PAGE . Alignments with the vaccinia virus D1 subunit N7-MTase (VV-D1) and other confirmed N7-MTases reveals several conserved domains, however the the natural N- and C-terminal borders, and importance of these regions for activity was unclear. As such, several lengths of the putative MTase, ranging from 260 to 336 amino acids in length were expressed and purified. All expressed domains include the five conserved motifs identified by structural alignment with the vaccinia virus D1 subunit N7-MTase (VV-D1). High yields of soluble protein (∼20–30 mg/l culture) can be obtained from a 311 amino acid domain spanning residues Ala) Figure . Gene coE Figure , suggest3H-AdoMet as the methyl donor. High levels of methyltransfer are observed for three out of the four soluble proteins, with no activity for controls lacking either RNA or protein  of WBV-295 are functionally important. These results confirm that the coding sequence encompassed by Asp1374–Ile1668 in the ORF1a of WBV harbors methyltransferase activity.The soluble protein products were tested by filter binding assays (FBA) for their ability to methylate a short (13-mer) GpppG-capped RNA substrate using radioactive n Figure . WBV-29513, mGpppN, GpppNm, mGpppNm) . The first nucleotide of the WBV genome is a G, however both GpppG and GpppA capped RNA substrates are methylated equally well  Figure . High lel Figure . Likewise Figure . These r viruses ,53. To cn Figure . No chanthe case . We therO methylation is pH-dependent ) are highly methylated, confirming that this MTase does not target internal residues  yielded a clean product of expected molecular weight, possessing activity levels equivalent to WBV-295 . A C-terminally truncated construct , with an alternating βαβ structural motif, characteristic of the class I family of MTases. This core structure is formed by a central, seven-stranded β-sheet , flanked by α-helices on each side . The final β-strand (β10) is inserted in an anti-parallel orientation between the fifth and seventh strands (β5 and β9) of the β-sheet. In addition to this central fold, two other structural features are present; a flexible ‘3β-flap’ domain comprised of a short α-helical loop followed by three anti-parallel β-strands (β1 and β6–β8) and an α-helical bundle  wrapping around α1, herein referred to as the α-bundle region  , VV-D1  and Hcm1 from humans  were additionally identified as potential homologues  as the closest homolog, with 16% sequence identity ( Å rmsd) . Despitemologues . SuperimThe first 22 residues of the WBV N7-MT-1a are unresolved in the structure. This region is important for the methylation activity, as the WBV-280 construct (lacking residues Asp1 to Ser16) is > 100-fold less active than its longer counterpart, WBV-295 Figure . ComparaA comparison of the 3β-flap domains of these five MTases shows the WBV N7-MT-1a flap-structure is most similar to that of Ecm1, which is also formed by an α-helix and three-stranded antiparallel β-sheet . NeverthThe α-bundle domain is the most structurally diverse region of the MTase. While all related MTases possess a similar domain between β9 and β10 of the central β-sheet, the length, position, and number of α-helices vary substantially between these structures. The human N7-MTase contains an additional insertion in the α-bundle region (referred to as the lobe), which is absent in other structures, including WBV N7-MT-1a. The observed variability of the 3β-flap and α-bundle region is indicative of their dynamic role in the accommodation and stabilization of the RNA GpppG-cap structure during cap 0 synthesis and as such are structural markers of the N7-MTase fold.A sequence homology search using the structural alignment of the five N7-MTases retrieved a pool of 120 diverse sequences, of which 6 conserved sequence motifs involved either in AdoMet binding and catalytic transfer  or in the GpppG-cap structure binding  can be defined  located in the β1–α2 loop, that accommodates AdoMet binding. In contrast, for the VV-D1 MTase the adenosine nucleoside is bound in the rarer syn-conformation, likely due to the side chain of an aspartic acid (D598), that clashes with the adenosine base in the anti-conformation . The site is an open, moderately charged pocket, lined on one side by residues from α1 and α9 of the α-bundle  and by analogy with the Ecm1 structure, complexed with e Figure . The floe Figure , C. While Figure . The onlTobaniviridae family members. This family is composed of 23 members which infect a range of mammalian  and non-mammalian (various fish and reptiles) hosts. Remarkably, the N7-MT-1a domain appears to be present only in the 18 non-mammalian Tobaniviridae members . These viruses currently fall under the subfamilies Serpentovirinae and Piscanivirinae. While there is still some ambiguity for the precise location of N- and C-terminal domains, the characteristic secondary elements of RF enzymes (βaβ-fold) and sequence motifs could be easily identified for all of these viruses. We conclude that N7-MT-1a is a genetic marker for non-mammalian Tobaniviridae.Using the newly defined WBV N7-MT-1a domain, we performed a MSA to look for the presence of this enzyme in other Picornaviridae and Caliciviridae families do not possess any capping enzymes, but rather covalently attach a small viral protein (known as the VpG) to the 5′ end of the genome which functions as a cap-substitute (O structure (m7GpppN)  . In cont RNA cap ,60–64. GCoroniviridae, the cap N7- and 2′O- methyltransferases have been well-characterized, residing in nsp14 and nsp16 of pp1ab, respectively  failing to converge toward either a consensus model or a partial model suitable for phasing the data. In terms of sequence, structure and substrate-specificity, there is no homologous MTase currently identified for any other +RNA virus to date. It is also remarkably different from the ORF1b N7-MTase gene of CoVs, suggesting that these MTases were acquired through separate evolutionary events.For ectively ,19–21. Ntructure . In mostTobaniviridae family. Remarkably, this domain is only present in the 18 non-mammalian host Tobaniviridae members. The acquisition of such a critical gene is a considerably rare event. Combined with its unexpected genomic location in ORF1a, we believe that the presence of this MTase is significant enough to warrant its consideration as a genetic marker for this family of viruses. Consequently, we propose that the other four mammalian-infecting Tobanivirus members be reassigned to a new, separate nidovirus family.Using the newly defined WBV N7-MT-1a domain, we revisited the O MTase, which maintains its conserved genomic location at the end of ORF1b? While we cannot definitively answer these questions, we speculate that it is related to the reptilian/fish host specificity of these viruses. More specifically, it has been shown that there is an inverse correlation between levels of DNA methylation and body temperature. In reptiles and fishes , methylation levels are higher than those of warm-blooded mammals , potentially explaining the need for higher copy numbers of this enzyme. Both cellular and viral mRNAs have been shown to be internally methylated, most commonly at the mresponse . Here, wCellular MTases have additionally been shown to methylate various other RNA substrates, including transfer RNA (tRNA) at various bases and positions . A strucTherefore, while we cannot definitively rule out the possibility that the tobanivirus N7-MT-1a recognizes another RNA substrate, the lack of a N7-MTase in ORF1b, the structural homology with known N7-guanine RNA MTases, the absence of homology with other protein or tRNA MTases and the strong N7-guanine specific cap activity, substantiates that the WBV pp1a MTase is the missing N7-guanine MTase involved in the RNA capping pathway.Nidovirales genome. Remarkably, this MTase is different from the ORF1b N7-MTase present in Coronaviridae. In the group of large RNA viruses, these two MTases were therefore likely acquired during separate events. Its structure belongs to the Rossman-fold family of enzymes, making it the first N7-specific RF MTase identified for (+)RNA viruses. Furthermore, its genomic location in ORF1a may also reflect a necessity for higher levels of methylation in cold-blooded hosts, supported by the fact that this domain is missing in tobaniviruses infecting mammalian hosts. Based on this, we propose that the Tobaniviridae family be further refined using this enzyme as a genetic marker.In conclusion, we have demonstrated the presence of an N7-specific MTase domain in ORF1a of non-mammalian tobaniviruses, highlighting the amazing plasticity of the Tobaniviridae N7-MT-1a domain was deposited to UniProt to update accession numbers polyprotein 1a (Q008X5) or 1a-1b (Q008X6).Reference alignment corresponding to Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession number 7Z05 and 7Z2J.gkac876_Supplemental_FileClick here for additional data file.