These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5′ and 3′ UTRs is still in development.RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise high throughput, cost efficient short-read RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information—for example, the ones represented by the 10x Genomics linked read methodology. The shift toward long-read sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for  Furthermore, the complexity of the transcriptome is increased by the fact that most genes possess more than one transcriptional isoform (RNA sequencing (RNA-Seq) using short-read sequencing technologies currently offered by Illumina or Thermo Fisher (Ion Torrent) represents the standard and widely used method for transcriptome profiling . Recentl isoform . These i isoform . Reconst isoform . TherefoCurrently, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) provide the most widely used long-read sequencing technologies. Read lengths achieved with these technologies (∼15 kb for PacBio and > 30 kb for ONT) surpass lengths of most transcripts. A relatively high cost and other limitations discussed here have prevented the wide use of these technologies in RNA-Seq, but now lower cost is promised through high-throughput nanopore sequencing approaches (currently represented from the ONT GridION and PromethION instruments), as well as the next generation of PacBio Sequel instrument (Sequel 2). For example, the per base sequencing cost on the PromethION platform can be as low as half the corresponding one on the MinION platform. In addition, the portability of the MinION instrument enables the sequencing of virus derived cDNAs and the identification of pathogens in the field .TM (Switching Mechanism At RNA Termini) technology, commercially available from Clontech (United States), makes full length mRNA sequencing achievable with the added benefits of more accurate transcriptomic studies  The first approach concerns the quantification of already known gene and isoform models. (ii) The second approach deals with the quantification of already known gene or isoform models, as well as the quantification of transcripts derived from novel isoform models . In thisThe aim of the following paragraphs is to present full-length cDNA synthesis strategies for long RNA molecules. Total RNA includes unprocessed transcripts, processed transcripts, and degradation products. Although the processed transcripts are the largest fraction of RNA molecules, the long-read sequencing platforms have been used to investigate all the different classes of RNA molecules. Below, we will first present the properties of the long-read RNA molecules that can be used to differentiate between the different categories. Next, we will present the library preparation methods that are exploiting these properties to specifically enrich for the different categories.7G cap, the RNA molecules can be grouped in the following three categories  or on the 5′ and 3′ end moieties present at the end of the molecules. The 5′ moieties correspond to the chemical entities attached on the fifth carbon (5′-C) of the sugar ring of the first nucleotide . The 3′ moieties correspond to the chemical entities attached on the third carbon  or the second (2′-C) carbon of the sugar ring of the last nucleotide. Nucleotides that have different chemical moieties on the second carbon of the sugar ring, except the hydroxyl group, are part of the group of modified bases. Depending on the chemical moieties of the fifth carbon of the sugar ring of the first nucleotide and on the chemical moieties of the third carbon of the sugar ring of the last nucleotide as well as the presence or not of a poly-A tail and of the mtegories .7G) RNA molecules. In this category, the RNA molecules correspond to full-length molecules (poly-A and non-poly-A molecules). Additionally, in this category, we can find RNA fragments that derive from either 3′ exonucleolytic degradation or endonucleolytic degradation of full-length RNA molecules. 3′–5′ exonucleolytic cleavage can occur, for example, from the Rrp44 ribonuclease of the exosome complex  The7G of the mature mRNA molecules prevents the ligation of adaptors on the 5′ end by the T4 RNA ligase 1. The T4 RNA ligase 1 can add an RNA adaptor only in the presence of 5′-P RNA molecules and not in the presence of a 5′-OH. The truncated T4 RNA ligase 2 can add a ssDNA adaptor only in the presence of 3′-OH RNA molecules. The presence of 3′-P inhibits the ligation of the adaptor with the truncated T4 RNA ligase 2 enzyme. In other methods, the small amounts of RNAs bearing 5′-OH termini can be ligated to the 5′ adaptors by using a RtcB RNA ligase (NEB) . Then, the RNA molecules can be either isolated with the help of these probes bound on streptavidin beads, or these probes can be used to prime reverse transcription . Alternase (NEB)  (Supplemse (NEB) .To sequence a specific subpopulation of RNA molecules, we can use either a method that targets a specific feature of this population (feature selection method) or a method that targets a feature of the non-targeted population which will eventually exclude it from the pool of RNAs (feature exclusion method). An example of the feature selection method is the standard reverse transcription from a poly-T primer. In this case, molecules with a poly-A tail can be specifically enriched with a poly-T reverse transcription primer and a Template Switching protocol, with a reverse transcriptase that shows a capped-dependent terminal transferase activity  into the same consensus molecule . NeverthIn the case of the reverse transcription of the viral RNA genomes, another artifact can arise when more than three nucleotides of the MinION strand switching oligonucleotide are found on the viral genome, allowing both template switching and false priming. It has been shown that this can result in a large number of false-positive 5′ ends . TSS varBelow, we will initially present some characteristics of the long-read sequencing platforms followed by a description of the current platform specific library preparation methods. A summary of the information presented in the following sections, regarding the technical comparison between the PacBio and the ONT platforms, is provided in For a complementary review, see The first PacBio instrument to be launched was the RSII, followed recently by the Sequel, which carries several modifications and improvements of the original technology that result in higher throughput. The number of ZMWs increased from 300,000 microwells on the RSII system to 1,000,000 microwells (1M SMRT cell) on the Sequel and SMRT cells with 8,000,000 microwells are scheduled to be produced. In our experience, read lengths of 20 kb can be achieved, while individual reads can exceed 60 kb, and there have been reports of maximum polymerase read lengths of 92.7 kb .The PacBio systems have the limitation that are best suited to operate within dedicated facilities and are currently far away from being developed as bench top versions. The long-read lengths achieved with this technology, coupled with the Iso-Seq RNA sequencing protocol discussed below and downstream data analysis pipelines developed by PacBio provides a powerful approach to RNA analysis.Originally loading the prepared library on the flowcell revealed a bias toward most efficient loading on the ZMWs array of shorter molecules . This prPacBio provides “SMRT Analysis,” an open-source bioinformatics software suite, for the analysis of data from SMRT technology. Here, we will focus only in the analysis of RNA-Seq data. Due to the small size of RNA molecules and the circular nature of sequencing, the forward and reverse strand of the same molecules can be sequenced multiple times, and all of them exist sequentially in the same sequenced read. Each time one strand of the cDNA molecule is sequenced, we refer to this event as a “subpass” and the produced part of the sequenced read as “subread.” The complete analysis of PacBio RNA-Seq data can be divided into five main processes: read-of-insert generation and classification, clustering, polishing, alignment, and visualization. Read-of-insert (ROIs) generation involves determining the highest quality sequence for each ZMW microwell; this includes inserts with < 2 subpasses and circular-consensus sequence (CCS) reads generated from inserts with ≥ 2 full-pass subreads. In the first case, the highest quality read is the longest subread or the only subread available. In the second, case a consensus is created from the full-length subreads. The ROI is then classified into “full-length” (FL) if the PacBio adaptor sequences are detected in both the 3′ and 5′ ends of the subread/consensus along with a poly-A tail. Alternatively, the ROI is classified into “non-full-length” (NFL) if the PacBio adaptor sequences are absent from either the 3′ or 5′ end or from both ends. Subsequently, the FL reads are processed through the Iterative Clustering for Error Correction (ICE) algorithm to build consensus clusters of one type of molecules. Each FL read is assigned to only one cluster while each cluster  comprise of one or more FL reads. NFL reads are used to increase the coverage of each ICE consensus isoform. With enough FL and NFL coverage, the Quiver algorithm (for PacBio RS II data) or the Arrow algorithm (for Sequel data) polishes consensus sequences yielding high-quality (HQ)  and low-quality  sequences depending on the number of reads present in each cluster . FinallyWith the Sequel system v6.0 release and the sequencing chemistry v3.0, a 1M SMRT cell can produce on average ∼615,000 sequenced reads, with a sequencing yield of ∼7.69 Gb and an average sequencing size of ∼12.5 kb from a 10-h sequencing movie time . The lenNanopore sequencing is a relatively new, single-molecule sequencing technology. Oxford Nanopore Technologies (ONT) has pioneered the development of nucleic acid sequencing using protein nanopores and commercialized its sequencing platform, the MinION, in 2014. The MinION is a miniaturized portable USB-powered device and the first commercially available protein nanopore sequencer. In contrast to all other commercially available sequencers to date, ONT technology determines the sequence of nucleic acids in a molecule directly without the need of amplification, sequencing by synthesis, or by ligation. A protein nanopore is inserted into a synthetic electrical resistant lipid membrane and immersed in an electrolyte solution. An electric field applied across the membrane drives DNA molecules through the nanopore while the current flow within the nanopore is recorded. The unique pattern of current disruption generated by the different nucleotides as the nucleic acid molecule is driven through the pore is used to determine the sequence of DNA. Nanopore sequencing technology currently generates the longest raw reads with no theoretical limits , a signiRNA sequencing with ONT can either be performed via a cDNA synthesis step or by direct RNA sequencing. The cDNA molecules are end repaired, dA-tailed, and then ligated on adaptors with dT overhangs. In contrast to the PacBio, where the polymerase is loaded after the addition of the adaptors, here the adaptors have pre-bound the molecular motor. The molecular motor will then drive the 5′ end strand inside the nanopore and the nucleic acid strand will be sensed by the platform  technologies have some error rate during basecalling. Depending on the application, even the short-read sequencing technologies may have an unacceptable high error rate, as in, for example, the detection and quantification of rare variants/low frequency mutations among the different genome copies in heterogeneous mixtures of cells or molecules. The raw read error rate of the basecalling informatics for the PacBio Sequel is ∼85% . In the 2 chemistry. The nature of the nanopore sequencing platform does not permit to sequence the same molecule more than two times. The 2D chemistry permitted the sequencing of the two individual strands of a DNA molecule through the introduction of a hairpin in one end of the DNA molecule. In 1D2 chemistry, the hairpin molecule was abandoned and a time-based inference of the two complimentary strands was adopted. Although both these chemistries are significantly different in the way the sense and antisense strand of the DNA molecule are sequenced, they both can increase the accuracy by 5–15%  and a sequencing adapter . The RTAThe primary limitation of this technology is the high input material requirements, currently around 500 ngs of polyA + RNA. These RNA input requirements are not physically achievable with most original clinical samples and further improvements of the platform might reduce the input material. Another limitation is that without the presence of an extra adapter sequence at the 5′ end of the RNA molecule, there is a loss of coverage at the extreme 5′ end of the molecule, which is due to unreliable processivity as the last bases of the molecule pass through the pore, resulting in the loss of 10–15 nucleotides not being sequenced . Along tH. sapiens  and of the synthesized cDNA from each cell with a cell-specific barcode. Due to the high error rate of the long-read sequencing platforms, the PacBio CCS reads have been used to more confidently sequence either the UMIs or the cell-specific barcodes.Demultiplexing of sequenced reads with UMIs can lead to better quantification of transcripts in single cells . As the Single-cell barcode identification was only performed after combining the 10x Genomics Chromium platform (abbreviated below as 10x) with PacBio in a method called ScISOr-Seq . With thSimilar with the approach above, single-cell cDNA synthesis with 10x has been combined with transcriptome profiling on the nanopore platform . For theA similar single-cell experiment was performed where the authors were trying to identify hypermutations on B-cell receptors . The difIn some of the above studies, the rational of using a long-read sequencing approach over a short-read sequencing one, for isoform identification and quantification, has already been commented in a recent review . From ouThe rest of the authors of the studies presented in this paragraph, additionally to gene expression quantification, they tried to quantify the different isoforms present in the single cells. Given that the isoform abundance reconstruction from full length short-read sequencing cDNA data can only give confident observations on differential exon abundance and not on the expression changes of the full length reconstructed isoforms, the authors correctly selected the full-length long-read sequencing approach to answer their biological question. For example, Moving away from gene or isoform abundance quantification, another study presented in this paragraph used the long-read sequencing data to perform cell clonality studies. For example, Short-read and long-read sequencing technologies have obvious differences in read lengths and throughput and yet the number of reads is usually a critical factor in RNA-Seq applications and cost. Standards and expectations have already been established for short-read technologies. For example, for the accurate quantification of highly abundant genes (FPKM > 10) 36 million short-reads are needed whereas for the accurate quantification of low abundant genes (FPKM < 10), the corresponding sequencing depth can go up to 80 million short-reads . As expeThe number of molecules sequenced in PacBio is always proportional to the number of microwells present in the SMRT cell. As the rate of occupancy of the microwells follows a poisson distribution, there is always care to avoid overpopulating the SMRT cell with molecules that can lead to occupancy of the ZMW microwell from more than two molecules and a sequencing read that will either be badly basecalled or will have a random basecalled sequence. This means that trying to saturate the SMRT cell will significantly increase this problem. Re-engineering the ZMW microwells by introducing nanopores at the bottom has been proposed to alleviate this problem and offer a complete saturation of the SMRT cell . NeverthDroplet-based microfluidic approaches can be applied to generate sequence information from individual full-length cDNA molecules through the development of the spISO-seq method , which iDespite the limitations, the advantage of these methods  is the lThe last five years have seen a dramatic improvement in the nanopore technology. Both the accuracy and the yield of the ONT MinION platform have significantly increased. In parallel alternative versions of the nanopore platform have been created  that promise a higher parallelization of sequencing, which can offer higher yield. The PacBio platform is also preparing major improvements like the introduction of 8 million ZMW well SMRT cells. Given the increased yields there is a considerable cost reduction in the experimental design that can be accompanied with sample barcoding possibilities. One important advantage of the MinION is that the signal processing and the basecalling can be done in a laptop without the requirement of a server cluster, a feature that has proven its utility in field applications. Overall, the long-read sequencing platforms have been used for a variety of applications that mainly complemented short read approaches, but now, single molecule, long-read sequencing approaches will become a mainstream approach in RNA-Seq.SO, AB, SF, and JR wrote the manuscript. SO, AB, HD, and PB prepared the figures. All authors discussed the final manuscript.JR is a member of the MinION Access Program (MAP) and has received free-of-charge flow cells and sequencing kits from Oxford Nanopore Technologies for other projects. JR has had no other financial support from ONT. AB has received re-imbursement for travel costs associated with attending Nanopore Community meeting 2018, a meeting organized by Oxford Nanopore Technologies.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is quantitative reverse transcription polymerase chain reaction (qRT-PCR), yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito ribonucleic acid (RNA). In this paper, we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing confident detection.in silico analysis of both viral and mosquito  genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedes genomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes. Standard curves constructed with in vitro transcribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay.Our new oligo design to detect ZIKV RNA included Aedes aegypti immune response to virus infection.Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegypti presented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.The experimental approach to detect ZIKV RNA in  Flavivirus of the Flaviviridae family. ZIKV infection in humans usually results in mild disease or asymptomatic infections; however, it can develop into to severe symptoms that can be lethal. The symptomatology can include fever, rash, arthritis and/or arthralgia and/or myalgia, conjunctivitis and fatigue. Neurological complications caused by ZIKV infection were reported in adults (Guillain–Barré syndrome) and neonates  [Zika virus (ZIKV) was first isolated in Uganda, from a sentinel rhesus macaque in 1947 [cephaly) .Before a major outbreak of Zika cases in 2007 at the Pacific Island of Yap in the Federate States of Micronesia , ZIKV inAedes aegypti and Aedes albopictus. In the current state of globalization and climate change, the frequency of human disease outbreaks related to arboviruses, including Zika, has increased in urban centers with competent vectors [ZIKV is transmitted to humans by the bite of an infected mosquito. The main vectors associated with transmission in the urban cycle are  vectors –13, reveMolecular detection of ZIKV ribonucleic acid (RNA) in mosquitoes can be challenging due to the limited number of primers and probes published –27, as wIn recent studies utilizing RT-qPCR to detect ZIKV RNA in mosquitoes, which mostly analyzed vector competence, it is possible to observe a methodological trend favoring the utilization of hydrolysis probes as a fluorescent label (83% of the papers in literature) and the use of viral RNA extraction kits to obtain the RNA templates (56% of the studies). However, the number of studies could actually be higher since some of them do not specify the extraction RNA method used for the experiments , 31–51. Ae. aegypti. In order to avoid nonspecific ZIKV RNA detection due to possible viral integration in the mosquito genome, in silico analysis of the Ae. aegypti, Ae. albopictus and ZIKV genomes were conducted to find sequences conserved between Asian and African ZIKV phylogenetic lineages [Aedes spp. genomes. Primers were designed to detect ZIKV RNA using these determined target regions. Primers were tested on in vitro transcribed ZIKV RNA as well as RNA samples from mosquitoes infected with ZIKV and the positive mosquito samples were used for transcriptional level analysis of Defensin A, an antimicrobial peptide (AMP) involved in Ae. aegypti immune response [In this study, we present a one-step qRT-PCR protocol that both detects ZIKV RNA and can be used to evaluate gene expression from the same sample of infected lineages , 58 but response , 60.2 in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum  and 1% penicillin-streptomycin . Infected cells were propagated for 5–7 days before supernatant collection and/or RNA extraction.The Vero (ATCC CCL-81) cell line was used for growing ZIKV Puerto Rico-PRVABC59  and MR766  strains. Cells were grown at 37 °C and 5% COAedes aegypti (Rockefeller strain) were used in all experiments. Mosquito colony was maintained in a secure insectary . The mosquitoes were bred and maintained in a controlled atmosphere . Larvae were fed with powdered fish food  and adult mosquitoes had access to 10% sucrose solution ad libitum. Female mosquitoes (7–14 days-old) were infected by blood-feeding using a Hemotek with ZIKV infected cell supernatant mixed with serum-inactivated human blood from healthy donors , in a 1:1 proportion. The final titer for ZIKV PRVABC59 strain, in the blood solution, was 4 × 106 plaque-forming unit (PFU)/ml. Mosquitoes were allowed to blood feed for 30 min, then ice-anesthetized and non-engorged females were removed. At 7 days post-infection, the fed mosquito whole bodies were homogenized in lysis buffer  supplemented with β-mercaptoethanol (10 µl/ml) and stored at − 20 °C until RNA extraction.https://www.ebi.ac.uk/Tools/msa/clustalo/). In addition, an alignment of the ZIKV NS5 sequence  was performed with the Ae. aegypti and Ae. albopictus sequences from “EST”, “Assembled transcriptome” and “Transcripts” datasets of the VectorBase Bioinformatics Resource (https://www.vectorbase.org/). A highly conserved sequence region between analyzed ZIKV strains, but divergent from Ae. aegypti and Ae. albopictus mosquito genomes, was identified , the characteristics of the designed primers (NS5-2362F and NS5-2457R) are demonstrated on Table The ZIKV Nonstructural protein 5 (NS5), the polymerase used for viral RNA synthesis , was theied Fig. . This seAe. aegypti using RNeasy Mini kit (Qiagen) according to manufacturer’s instructions, which included the use of the cell lysate biopolymer-shredding system  and on-column deoxyribonuclease (DNase) digestion using RNase-free DNase I (Qiagen). RNA sample concentrations were measured using NanoDrop Spectrophotometer (Thermo Fisher Scientific). For ZIKV RNA detection, one-step RT-qPCR assays were performed on a CFX96 Touch Real-Time PCR Detection System , using QuantiFast® SYBR® Green RT-PCR or QuantiFast® Probe RT-PCR kits (Qiagen) according to manufacturer’s instructions. Primers and probes used on RT-qPCR reactions are shown in Table in vitro transcribed RNA (as described below). Aedes aegypti Actin was used as control to confirm RNA integrity of samples submitted as well as for absolute quantification and normalization of infection results. Amplicon specificity of ZIKV NS5 was evaluated by the melting curve peak (80 ± 0.5 °C). The specificity of ZIKV NS5 primers was also evaluated by testing them on viral RNA of dengue virus (DENV-2 NGC strain) obtained from infected Vero cells and Ae. aegypti mosquitoes, as negative controls. The relative expression level of Ae. aegypti Defensin A was calculated by DeltaDelta CT (cycle threshold) method, using Ae. aegypti Actin as a gene reference. The primers utilized to amplify Defensin A are shown in Table RNA was extracted from infected cells and/or in vitro transcription using the Megascript kit , according to manufacturers’ instructions. The number of single strand RNA copies (molecules/μl) was calculated as described by Faye et al. [Five µg of RNA from Vero cells infected with ZIKV MR766 or PRVABC59 strain were used for synthesis of cDNA using SuperScript III Ribonuclease (RNase) H-Reverse Transcriptase . First strand cDNA was subjected to PCR using DreamTaq deoxyribonucleic acid (DNA) Polymerase (Thermo Fisher Scientific) with ZIKV 1086-T7/ZIKV 1162c or NS5-2362-T7 F/NS5-2457R primer sets . At that point, PRVABC59 NS5 sequences without similarity to mosquito genomic sequences were aligned with three ZIKV strains from the Asian phylogenetic lineage  and 2 ZIKV strains from African phylogenetic lineage  using ClustalW. At the conclusion of the analysis, a region of 96 nucleotides from ZIKV NS5, highly conserved between ZIKV strains but divergent from Ae. aegypti and Ae. albopictus, was identified and utilized as template to design qRT-PCR primers . The identities found in four of many significant alignments obtained between the primers annealing sites and mRNA sequences of Ae. aegypti are demonstrated in Fig. Once ZIKV 1086/ZIKV 1162c primers were designed to detect ZIKV RNA in human samples, the observed nonspecific amplifications could be caused by similarities between mosquito and viral sequences. Then, we performed the alignment of the sequence amplified by ZIKV 1086/ZIKV 1162c primers and in vitro, were used to create standard curves to assess the limit of detection, efficiency and reproducibility of the RT-qPCR assay using NS5-2362F and NS5-2457R primers and SYBR Green as fluorescent dye. The gold standard used in the literature (ZIKV 1086/ZIKV 1162c primers and 1107-FAM as the dye) was used in comparisons; referred to in the paper as “1107-FAM probe”. The detection of the RT-qPCR reactions was linear over six 10-fold dilutions (109 to 104 copies/reaction) using synthetic RNA with sequences from the MR766 and PRVABC59 strains  from synthetic RNA standard curves was calculated to assess the efficiency of reactions using NS5-2362F and NS5-2457R primers, compared to the gold standard (1107-FAM probe). The results show that reactions using the NS5 primers are highly efficient (R2 = 0.99) in the detection of RNA sequences from both ZIKV strains tested (MR766 and PRVABC59)  were calculated from quantification cycle (Cq) values obtained using 10The data shown in Figs. Aedes mosquitoes. Total RNA from ZIKV-infected whole mosquitoes (PRVABC59 strain) was extracted 7 days post-blood-feeding. RT-qPCR using the NS5-2362F and NS5-2457R primers showed an infection rate of approximately 47% , with infection levels of 9.9 × 104 ± 8.1 × 104 ZIKV RNA copies/ng total RNA. The mosquito samples were also analyzed using the 1107-FAM (gold standard) and the results demonstrate a highly approximate ZIKV RNA detection level , as well as the same infection rate, with that obtained using NS5 primers  female Ae. aegypti antimicrobial peptide Defensin A has a role in the mosquito immune response to dengue, Chikungunya virus and Zika virus infection [Since the nfection , 65, we nfection  that demAe. aegypti, using one-step qRT-PCR and oligos based on the viral NS5 gene sequence. This assay also uniquely allows for the analysis of transcriptional levels of mosquito genes during ZIKV infection from the same samples. The method of ZIKV RNA detection in mosquitoes presented here is based on RT-qPCR using a standard RNA extraction kit, primers designed to avoid nonspecific amplification of the mosquito genome and SYBR green as a fluorescent label. Our protocol is relevant since the gold standard for the detection of ZIKV by qRT-PCR in the literature is based on viral RNA extraction kits and the use of hydrolysis probes as the fluorescent label, which is relatively effective but expensive, and the published primer and probe sets were optimized in assays using mammalian samples.With increasing outbreaks of ZIKV in humans globally and subsequent increase in experimental analysis of viral pathogenesis and interactions in the mosquito vector, an effective and reliable method to detect ZIKV infection in mosquito samples is essential. This study has presented a sensitive, efficient and reproducible assay for the detection of ZIKV RNA in In recent studies that used qRT-PCR analysis to detect ZIKV RNA in mosquitoes, most used hydrolysis probes as fluorescent labels. More than half of the studies that used fluorescent probes to detect ZIKV RNA also used viral extraction kits and four did not describe how RNA extraction was performed , 48, 49.To date, only five studies , 43, 47 In summary, from recent studies in which qRT-PCR was used to detect ZIKV RNA in mosquitoes, 83% of studies used hydrolysis probes as fluorescent labels and 56% describe use of viral RNA extraction kits (considering studies that do not specify extraction RNA method it can reach 70%). Although this approach has been demonstrated to be effective, it is also expensive. In addition, since the isolation of viral RNA is prioritized, this method does not permit the evaluation of the expression of mosquito genes during ZIKV infection. Studies that analyze ZIKV infection in mosquitoes, for example vector competence analyses, often require the assessment of different tissues at different time-points from individual mosquitoes. This work generates large numbers of samples, each requiring RNA extraction as well as multiple qRT-PCR reactions for sufficient analysis, and all of which using current methods come at great expense. Due to this financial barrier, the method presented in this study is an especially relevant alternative for the inexpensive, rapid and reliable detection of ZIKV in mosquito samples by qRT-PCR.Considering that majority of ZIKV outbreaks occur in developing countries, the availability of highly sensitive, but also affordable, assays to detect ZIKV can greatly enhance local vector surveillance and disease control. Taking into account the experimental conditions and reagents utilized in this work, the prices of PCR reactions using NS5 primers were 31% less expensive than using 1107-FAM probe .Ae. aegypti presented here has been demonstrated to be sensitive, efficient and reproducible for in vitro analysis in the laboratory as well as on infected mosquito samples. In addition, our presented protocol allows for the analysis of mosquito gene expression during ZIKV infection in the same samples and is a much less expensive approach optimized to minimize nonspecific amplification in Aedes spp. samples.The experimental approach developed and demonstrated in this study can significantly contribute to research efforts in study of mosquito gene expression during ZIKV infection. The assay used to detect ZIKV RNA in


The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the “gold standard” test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the “gold-standard” CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing. A scalable, high-throughput platform will be required to deliver millions of tests per day. Here we investigate recent advances and approaches to nucleic-acid testing for COVID-19. We highlight some findings from research groups who have compared commercial reagents or created homemade solutions in order to decrease cost or reliance on particular commercial reagents. We also outline several alternative nucleic-acid tests involving isothermal amplification or CRISPR-based detection. Finally, we examine some recent applications of specialized techniques such as sequencing, digital PCR, and DNA nanoswitches as tools for COVID-19 detection. We have tried to be as exhaustive as possible throughout this review, but due to the rapid daily developments in testing we may have unwittingly excluded some published works. Another review by On March 11th, 2020, the World Health Organization deemed COVID-19 a global pandemic . As of ARNaseP, are also included as a positive control for swab collection, RNA extraction and amplification. A spike-in control RNA, such as MS2 bacteriophage genomic RNA, may alternatively be used. Amplified products can be detected using TaqMan probe fluorescence or DNA-intercalating dyes, and a threshold cycle of amplification is set to distinguish positive from negative results. A test result is typically considered positive if amplification is observed for two or more viral targets, while it is considered negative if amplification is observed for the control RNA but for none of the viral targets . Specialized reagents or equipment can lead to high per-test costs and may limit the number of tests that can be conducted, in some cases resulting in a lag of several days before a patient receives a diagnosis. The variety of approaches presented here span a wide range of costs and processing times, with several published protocols reaching results in less than 1 h . Others have proposed completely novel solutions that can cut the testing time to tens of minutes but may still require costly reagents to perform. While widespread testing will necessarily require high-throughput approaches, other tests may offer higher sensitivity for low titer cases or rapid turnaround for point-of-care diagnosis. Recent ingenuity in COVID-19 nucleic-acid testing offers a wide range of solutions and further innovation may be required to maximize testing accuracy while providing a low-cost and fast-turnaround solution.The standard CDC RT-PCR test takes about 3 h to perform and costs ∼$10 per test . These samples are lysed and viral RNA is typically purified using either RNA extraction columns or magnetic beads . One adv) in VTM spiked directly into the RT-PCR master mix without an RNA extraction step , Buffer RLT (Qiagen), and MagNA Pure External Lysis Buffer (Roche). However, multiple researchers have recently found that when compared to commercial solutions, homemade solutions containing 4M  or 5M A guanidinion step . Merindoion step .Many groups are further optimizing direct-to-test addition by heating and/or lysing swab samples prior to testing. In one study, direct addition of swab samples in viral transport media to the Luna Universal Probe One Step RT qPCR master mix (New England Biolabs) accurately identified 92% of 155 COVID-19 cases but reached the detection threshold four cycles later  than a test using RNA extracted from a swab sample using the QIAamp Viral RNA Mini kit (Qiagen) . This prSlightly more complex approaches use lysis buffers to aid RNA recovery and improve RT-qPCR test sensitivity. In one report, positive patient swab samples diluted 1:1 into Quick Extract DNA extraction solution (a buffer containing detergents and proteinase K), heat inactivated and directly added to the RT-PCR reaction mix were detected at the same amplification cycle as, or even slightly before, samples processed with the QIAmp Viral RNA Miniprep kit (Qiagen) . AnotherThe discrepancy between the sensitivities of direct-to-test addition procedures may be due to differences in protocols and kits used for the RT-qPCR test and isolation of control RNA, types of lysis buffers, heating parameters, and varying viral RNA loads in the swab samples of each study. Despite these discrepancies, it appears that direct-to-test addition of a small volume of swab sample treated with lysis buffer or Proteinase K allows for robust SARS-CoV-2 RNA detection. Direct-to-test addition of patient swab samples may prove useful in settings where there is a lack of RNA purification reagents or time constraints that render laborious RNA isolations infeasible. Further work is required to ascertain optimal swab sample lysis, heating and storage conditions prior to direct-to test addition, as well as whether direct-to-test addition could be used in tests other than RT-qPCR.Ct) loss of synthesized SARS-CoV-2 viral N gene RNA after column purification. We found similar results with inactivated positive patient swab samples; Ct values were consistently lower when RNA was purified via isopropanol precipitation or using the Direct-zol RNA Miniprep Plus kit (Zymo Research) when compared to the RNeasy Mini Kit (Qiagen). However, we have not compared directly with the CDC recommended Qiagen QIAmp Viral RNA kit . Several recent papers have investigated alternative methods for RNA purification, including unique approaches as well as traditional laboratory techniques. N gene RNA resulted in almost no loss of RNA, with or without the presence of additional human RNA as a carrier . Standard laboratory RNA purification methods offer an attractive alternative to commercial kits, as they generally use inexpensive, abundant materials. For clinical testing, however, these solutions may be difficult to scale to high-throughput pipelines and may require special handling of hazardous materials. It may be useful to assess where RNA extraction can be eliminated while maintaining the necessary sensitivity and accuracy of testing. If eliminating RNA purification is not possible, however, these procedures could be useful as cheap, homemade solutions for small-scale testing operations.As uncovered by multiple groups, eliminating RNA isolation prior to RT-PCR altogether may be possible. However, a dedicated RNA isolation step may improve detection sensitivity or be necessary to remove incompatible sample buffers prior to amplification for some protocols. However, column-based kits used to purify RNA from the patient swab sample can also occasionally lead to unintentional carryover of ethanol or retention of some RNA, which can be kit-specific. In our laboratory, we have found that the RNeasy Mini Kit (Qiagen) leads to an approximately eightfold  and recombinase polymerase amplification (RPA) . LAMP usA slightly more recent addition to the isothermal amplification toolkit is RPA. RPA uses a recombinase to catalyze strand invasion of a primer into dsDNA. Single-stranded binding proteins are included to stabilize the open duplex structure and a strand-displacing DNA polymerase extends the primer . Some grSeveral groups have now developed novel isothermal protocols for detection of SARS-COV-2 RNA. A unique group of Cas nucleases, including Cas12 and Cas13 were recently discovered to have promiscuous DNA or RNA cleavage activities , which hCas13a is a nonspecific RNase that remains inactive until it binds its programmed RNA target. It has been harnessed for sensitive DNA or RNA detection in a method termed SHERLOCK  In SHERLCas12 is another member of the CRISPR-Cas effector family. It is an RNA-guided DNase that indiscriminately cleaves ssDNA upon binding its target sequence. In a method termed “DETECTR,” Cas12a ssDNase activation is combined with isothermal amplification to achieve sensitive and specific DNA detection . MultiplIn combination with fast isothermal amplification, CRISPR-based techniques can harness highly specific nucleases to achieve fast read-outs and sensitivity down to a few viral RNA copies. CRISPR detection can be coupled to lateral flow readouts, which are an attractive option for easy, at-home testing scenarios.Sequencing-based detection methods provide the benefit of collecting base-pair-level information of patient strains, which allows for viral mutation tracing but comes at the cost of expensive sequencing platforms and lengthy sample processing times. However, several laboratories have investigated high-throughput approaches or portable, fast sequencing to use this technology as a diagnostic tool for COVID-19. Nanopore target sequencing (NTS) is an attractive option for clinical testing because it is fast, highly portable, and sensitive. Wang et al. have developed an NTS approach targeting 11 viral regions that is able to detect as few as 10 viral copies/mL with 1 h of sequencing. By relying on a sequencing-based approach, this group also demonstrated that viral genome mutations can be identified within the target regions, and that an additional panel of targets against common respiratory viruses can be included to detect co-infection . AdditioBeyond the approaches described above, ingenious methods are being developed for widespread, at-home, or point-of-care COVID-19 diagnostics. Most isothermal amplification steps require incubation at elevated temperatures around 60°C. To facilitate isothermal amplification at remote testing facilities, While samples collected from patients with symptoms or who have been hospitalized seem to present relatively high viral titers that are likely to be easier to detect , testingParticularly as testing becomes more widespread, testing of the general population and asymptomatic individuals may lead to a large number of negative samples and a huge increase in the demand of testing supplies. In an innovative effort to further conserve resources using existing testing methodologies, some groups have investigated pooling many patient samples to decrease the number of tests required for larger populations. Proposed pooling approaches can be adaptive, where samples are first pooled and tested and positive pools are retested individually. This is a relatively simple solution which decreases overall testing resources used but may introduce several disadvantages, including longer wait times for results since positive samples must be iteratively tested, and a slight loss in sensitivity from diluting positive patient samples with negative ones. Multiple groups have modeled patient pooling and proposed algorithms that optimize positive sample detection and testing efficiency . Some si5 Zika RNA genomes per test for reliable detection and is thus far less sensitive than RT-PCR (Lastly, it is important to consider that enzyme-based tests are not always feasible in resource-limited scenarios. One potential alternative lies in the use of DNA “nanoswitch”-based tests that have been developed for Zika virus detection. Based on a DNA origami design, these nanoswitch DNA oligomers bind viral RNA to undergo a conformational change that can be visualized on an agarose gel. DNA nanoswitches targeting different species of RNA viruses can also be combined in one test, allowing for the detection of co-infections. Unfortunately, the Zika nanoswitch test requires ∼5.2 × 10n RT-PCR . Due to n RT-PCR . FurtherIn response to the tremendous global toll of COVID-19, researchers have rapidly mobilized to investigate solutions for testing, diagnosis, and treatment. Preprint and published articles from the past several months describe a variety of options for rapid, affordable, sensitive, and high-throughput nucleic acid testing, which is currently the most reliable approach for early detection of SARS-CoV-2. To address the dire need for increased testing, researchers across disciplines have quickly compared widely available commercial products, proposed repurposing existing reagents and infrastructure, and created novel laboratory solutions to optimize the COVID-19 testing pipeline. Academic researchers at a few institutions around the world have published detailed blueprints for establishing local pop-up testing centers, and others are likely to follow . A combiRT-PCR—reverse transcription polymerase chain reaction; amplification of RNA in a one-step reaction containing a reverse transcriptase enzyme, DNA polymerase, and a specific primer complementary to a target region.LAMP—loop-mediated isothermal amplification that uses four primers that recognize six complementary regions within the target. Amplification occurs from a strand-displacing polymerase and elongation of primers with self-complimentary regions for hairpins that prime further rounds of amplification forming large “cauliflower” structures of amplified products.RPA—rapid amplification; recombinase polymerase amplification uses a recombinase-primer complex which finds matches in the DNA or RNA template and enables strand exchange to form an open complex. Single-stranded binding proteins stabilize the open duplex and strand-displacing DNA polymerase amplifies the template.RAMP—a two-stage isothermal amplification technique combining a primary RPA reaction using the outside LAMP primers with a secondary LAMP reaction including self-complimentary internal primers.Ct or Cq value—in qPCR, the amplification cycle where the fluorescence curve exhibits the greatest curvature and exceeds the background fluorescence threshold.Sensitivity —the abilSpecificity —the abilAccuracy —the abilSupplemental material is available for this article.


Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding. The retroviral genomic RNA typically represents less than 1% of the total RNA in the infected cell but it is specifically packaged into new virions. The region of the viral RNA responsible for directed packaging into newly formed particles is called the packaging signal (Ψ). In HIV-1 it is an ~80 to 150 nucleotide segment located within the 5′ UTR of the viral genome . In the The main partner for the viral RNA in the packaging process is the retroviral protein Gag. HIV-1 Gag is a ~55 kDa polyprotein composed of several independently folded domains connected by flexible linkers. From the N- to C-terminus these domains are matrix (MA), capsid (CA), spacer peptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2), and p6 ), it alsIn vitro Gag-RNA binding experiments showed that in physiological salt concentrations, the affinity of Gag for Ψ and non-Ψ RNA is comparable. However, when binding is measured under conditions of higher salt concentrations which suppress nonspecific electrostatic interactions, preferential binding to Ψ RNA is revealed ,12. It hA previous SHAPE structure-probing analysis of the HIV-1 5′ UTR revealed that the main NC interaction domain within Ψ consists of seven short stretches containing unpaired guanosines that are recognized by the NC zinc fingers . It has However, the nature of the difference between Gag’s interaction with Ψ RNAs and non-Ψ RNAs is still unclear. Especially, how is it possible that the viral RNA is packaged into the newly formed virus almost exclusively? We have reported that Gag protein assembles much more efficiently upon interaction with the viral genomic RNA  than witIn this work, we analyzed which Gag domains and multimerization interfaces are important for binding to Ψ RNA and non-Ψ RNA. These experiments relied on microscale thermophoresis (MST) ,17, whicRNAs were produced by in vitro transcription of linearized plasmids containing the T7 promoter, as previously described . Ψ RNA iE. coli and purified following a previously published protocol [All the proteins were non-myristoylated and derived from WT Δp6 Gag (isolate BH10), expressed without any affinity tags in BL21(De3)pLysS protocol , followe8N Gag  has eighWM Gag  has trypSSHC Gag  has the Thermophoresis  measuremminF is unbound response, maxF is bound response, hill is Hill coefficient, and conc is concentration of the protein.All fitting of experimental data was performed using R (version 3.6.1) with installed mixtox package and binding equation:p < 0.05.The statistical significance of the differences between proteins binding to Ψ and to reverse complement RNAs was evaluated using two-tailed t-test with significance level 0.05. In 100 mM NaCl, the differences in binding were not statistically significant. In 300 mM NaCl, the differences in binding of 8N, HBR switch, and WM Gag were not significant. Proteins for which EC50 values could only be estimated, i.e., FW, NC 4A, NC 10A, and SSHC Gag were not evaluated for statistical significance. The differences in binding of WT, ΔMA, and Δ1 SP1 Gag in 300 mM NaCl were significant with Out of the six domains that comprise full-length Gag polyprotein, four of them are directly involved in Gag–Gag and/or Gag–RNA interactions during the virus particle assembly. They are MA, CA, SP1, and NC domains (reviewed in ). The NCTo address the question of which parts of the Gag polyprotein are important for selective packaging of viral RNA we prepared nine mutants of Gag that impair different regions known to participate in Gag–RNA or Gag–Gag interactions. The mutations can be divided into three groups. The first group has mutations in the MA domain. It is either a large deletion in MA (ΔMA Gag) or mutations altering the HBR of MA (8N Gag and HBR switch Gag). The second group contains mutants that have impaired the ability to form Gag dimers (WM Gag) or the six-helix bindle (Δ1 SP1 Gag). The last group has mutations in the NC domain affecting the zinc fingers (SSHC Gag), the charge of the NC domain (NC 4A Gag and NC 10A Gag), or the aromatic residues that contribute to RNA binding (FW Gag). The binding of the proteins to Ψ and reverse complement RNA was then tested in buffers containing near-physiological salt (100 mM NaCl) and at a higher salt concentration (300 mM); the resulting curves are shown in Therefore, we increased the salt concentration in the measuring buffer to 300 mM to reduce the nonspecific electrostatic interactions. Under these conditions, we were able to detect approximately 12 times stronger binding of WT Gag to Ψ RNA than to reverse complement RNA . If the basic amino acids in the HBR in the MA domain were mutated to asparagine (8N Gag) the affinity of Gag to RNA was decreased more than 25-fold and there was no selectivity towards Ψ RNA. A similar but weaker effect was observed with ΔMA Gag, which bound Ψ RNA approximately 3.5 times stronger than reverse complement RNA . SurprisDestroying the ability to form the six-helix bundle (Δ1 SP1 Gag) in Gag mildly affected the binding to RNA (two-fold difference in binding as compared with WT Gag). Interestingly, preventing Gag from forming dimers (WM Gag) decreased the affinity towards Ψ RNA 3.5 times but the affinity towards reverse complement RNA did not change significantly . This suWe were not able to detect any binding of NC 10A Gag and FW Gag with either of the RNAs and no binding of SSHC Gag and NC 4A Gag with reverse complement RNAs  in 0.3M Destroying the ability of Gag to coordinate zinc ions (SSHC Gag) led to an approximately 12-fold decrease in the binding affinity towards Ψ RNA and no detectable binding of reverse complement RNA. This result suggests that zinc fingers, while important, are not entirely responsible for the preferential binding of Ψ RNA. The lack of binding of NC 4A Gag to the reverse complement RNA, while partially preserving the ability to bind Ψ RNA, confirms the hypothesis that NC 4A Gag binds to Ψ RNA through non-electrostatic interactions that ensure the preferential binding of viral RNA . The absIn summary, the presented results demonstrate the importance of MA’s HBR for RNA binding and the supportive effect of Gag dimerization in selective binding of Ψ RNA. Although more experiments need to be performed to truly understand the underlying mechanism, these findings show the need to study Gag-RNA binding using constructs containing the MA domain and offer new evidence about the role of individual Gag domains in the initial stages of viral particle assembly.We present in vitro binding experiments of multiple HIV-1 Gag polyprotein mutants, with lesions affecting various Gag–Gag or Gag–RNA interaction interfaces, with RNA containing the Ψ sequence or with a control RNA. The results presented here show that the positively charged and aromatic residues in the NC domain are crucial for any RNA binding to occur. Strikingly, if these are present, then, the positively charged amino acids in the HBR of MA serve as an additional Gag-RNA binding interface that also contributes to the specific binding to Ψ RNA. The formation of the six-helix bundle and especially Gag dimerization contribute to tighter RNA binding even below the concentration needed for Gag polyprotein to dimerize in solution. This suggests that specific Gag–Gag interactions could be generated in the nascent Ψ RNA-Gag complexes.Taken together, the data help to identify the regions of Gag polyprotein that contribute to specific and nonspecific RNA binding. Our data confirmed that the most important determinants for Gag binding to RNA are the basic amino acid residues within the NC domain and the two aromatic amino acid residues in the NC zinc fingers. In high salt buffer, we were not able to detect any binding of FW Gag and NC 10A Gag to any RNA. On the basis of the highest protein concentration used in the experiments we estimated the EC50 values to be >30 µM. Therefore, we conclude that these two features are crucial for RNA binding.When the basic and aromatic residues were present, we observed binding to Ψ RNA even in the absence of zinc fingers (SSHC Gag) or with four basic amino acids between the zinc fingers neutralized (NC 4A Gag). The SSHC results are in accordance with previously published experiments ,12. The d for Gag dimerization is 5.5 µM [d for Gag–Gag interaction in free solution. In fact, since the Kd for Gag–RNA interactions is in the nanomolar realm while Gag–Gag interactions are in the micromolar realm, it is clear that binding nearby sites on an RNA molecule could lead to Gag–Gag interactions at far lower concentrations than can occur in solution. This could help explain how binding to RNA promotes particle assembly. It is striking that the multimerization mutants (WM and Δ1 SP1 Gag) affect binding to RNA because binding was measured at a protein concentration in which free WT Gag is monomeric. The Ks 5.5 µM . Somewhas 5.5 µM , who fouThe results of our binding experiments slightly differ from those published by Comas-Garcia et al.  which weLys3. It has been also reported that mutations in the HBR change Gag localization in cells in vivo [The role of the MA domain in interactions of Gag with RNA is complex. It has been reported  that a G in vivo . Our res in vivo  led to a


Today a new family of so-called “noncoding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5′ end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3′–5′ interaction have been reported, including m6A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m The potential of circular RNA to be translated has been studied since the 1970s. In 1979, an experiment was designed to determine the ability of circular mRNA to attach ribosomes . ConsequTen years later, the discovery of translation initiation mediated by internal ribosome entry sites (IRESs) broke the rule ,4,5. FurDespite these demonstrations, IRES function in cellular mRNAs remained questioned for a long time, although obvious in the case of picornaviruses whose genomic mRNAs are uncapped ,8. From 6-methyladenosine (m6A) [6A is a reversible epitranscriptomic modification found in many eukaryotic mRNAs [6A promotes cap-independent translation at sites called “m6A-induced ribosome engagement sites” (MIRESs) [CircRNAs may also be translated by another cap-independent mechanism mediated by the methylation of the nitrogen at position 6 in the adenosine base within mRNA, Nne (m6A) ,15. M6A ic mRNAs . When pr(MIRESs) . As IRES(MIRESs) ,17. CircIn parallel to the studies on covalently closed circular (CCC) RNA, a series of reports have shown that translation involves the functional circularization of mRNA. Already in the 1980s circular polysomes were observed by electron microscopy . It was Can we still consider an mRNA as linear? That is the question. This review article aims to propose an update of the data on covalently as well as noncovalently closed circular mRNA translation, as the landscape in the field has strongly evolved in the last years.The first RNA circles were observed in 1976 by electron microscopy in viroids (plant pathogens), then in 1979 in human HeLa cell cytoplasm ,25. MoreSry sex determination gene [Sry circular RNA. The authors made the assumption that it could have either a positive role by being translated by internal ribosome entry or a negative role as a noncoding RNA by preventing efficient translation [Sry circRNA has been shown to function as a sponge for the microRNA miR-138 with 16 putative sites for that miRNA [Sry circRNA thus indirectly acts on translation by preventing translational inhibition of miR-138 targets involved in activation of tumor cell growth and invasion [Shortly after, a circular transcript was identified after RNase H digestion of RNAs extracted from adult mouse testis as the most abundant transcript expressed from the ion gene . This trion gene ,28. A lonslation . When suat miRNA . Sry cirinvasion .Sry in 1993 turned out to be a general feature for thousands of genes in human and mouse tissue and in various cell types [In the last decade, the emergence of RNA deep sequencing technologies and of sharp bioinformatics analyses has generated a major leap forward in the field of circRNAs. The abundance of the circular transcript observed for ll types ,31. RNA-ll types . InitialCircRNAs exhibit different modes of action, depending on their composition which itself affects their localization. CircRNAs that contain intronic  or intronic plus exonic sequences (EIciRNAs) are nuclear and mainly regulate the expression of their parental gene. EIciRNAs have been shown to interact with RNA polymerase II subunits, with U1 snRNP and with the parental gene promoter where they behave as transcriptional enhancers . AnotherSry circRNA is a sponge for miR-138 [In parallel to the discovery of thousands of circRNAs expressed in mammalian cells, it has been established in 2013 that natural RNA circles exhibit the function of miRNA sponges: as mentioned above,  miR-138 . Another miR-138 . CiRS-7  miR-138 . The pri miR-138 ,38. Seve miR-138 . By prevkip mRNA by facilitating HuR translocation to the cytoplasm. As HuR is a negative ITAF able to inactivate the p27kip mRNA IRES, its presence in the cytoplasm would promote silencing of p27kip mRNA translation and prevent cell cycle inhibition by p27kip [In addition to being miRNA sponges, circRNAs are also sponges for RBPs. This has been shown for Foxo3 circular RNA that forms a ternary complex with cell division protein kinase 2 (CDK2) and its inhibitor p21, which blocks the CDK2 function and arrests cell cycle progression . RBP spoy p27kip ,44. Herey p27kip . In addiWe have seen above that circRNAs have a strong impact on translation as noncoding RNAs. However they can no longer be considered as noncoding RNAs due to the growing evidence that many of them are translated. In spite of the continuous controversy about the ability of cellular mRNAs to be translated independently of their 5′ end, the first demonstration of translation of a natural circular RNA was provided in 2014 for the covalently closed circle (CCC) 220 nt-length RNA of the virusoid associated with rice yellow mottle virus (RYMV) ,46. The muscleblind (mbl) locus that produces several highly expressed circRNAs and identified by mass spectrometry (MS) a 37kDa peptide produced by the circRNA CircMbl3. Furthermore, they identified IRESs in several circRNAs  by using the well-known bicistronic vector strategy and by measuring their resistance to 4E-BP overexpression (which inhibits cap-dependent translation) [The absolute proof of translation of cellular circRNAs was provided in 2017 by Pamudurti et al. [slation) ,17. Thisslation)   .In the same issue, Legnini et al. identified the translation of another circRNA, circ-ZNF609, expressed in murine and human myoblasts . A globaFollowing these two pioneer articles several additional studies have identified proteins expressed by circRNAs. Translation initiation always depends on IRESs, identified by the systematic use of bicistronic vectors. Zhang et al. revealed the existence of the circRNA circ-SHPRH whose expression is repressed in glioblastoma . Circ-SHA fourth coding circRNA, circβ-catenin, has been characterized in human cells and tissues . It is o6A-based ribosome entry sites, called MIRES [6A modification close to the start codon , suggesting MIRES-dependent initiation  identifying a higher density of m6A sites in the m6A methylome [6A sites present in the GFP circRNA are sufficient to promote reporter gene expression and that this translation is sensitive to the overexpression of the methylases METTL3/14 or of the fat mass and obesity-associated protein (FTO) demethylase. m6A-dependent translation initiation involves the initiation factors eIF4G2, eIF3A as well as the m6A reader YT521-B homology domain (YTHD) F3. The direct interaction of eIF4G2 with YTHDF3 suggested a possible role of the m6A reader in recruiting eIF4G2 to the m6A site, promoting by this way the internal entry of the translation machinery as eIF4G2 directly recruits eIF3 [6A methylation was also demonstrated in human embryonic stem cells by Zhou et al. who identified, by m6A-RIP, 1404 m6A circRNAs whose methylation is dependent on METTL3 [All circRNAs characterized above for translation contain an IRES. However an alternative to IRESs is provided by med MIRES . Supported MIRES . The autethylome ,53. Yangits eIF3 . In the n METTL3 .Very recently, Fan et al. generated a library of random 10-nt sequences inserted before the start codon of a circRNA-coded GFP and identified 97 IRES-like hexamer sequences enriched in thousands of endogenous circRNAs, whose pervasive translation is supported by mass spectrometry evidences . Several6A modification on the immune response [td autocatalytic intron from T4 bacteriophage and demonstrated that this self-spliced circRNA is highly immunogenic in mammalian cells [6A modification is the marker for self: they transfected DNA plasmids coding circRNAs generated by protein-assisted (circSELF) or autocatalytic splicing (circFOREIGN) into HeLa cells and found that circSELF but not circFOREIGN is associated with the m6A machinery [6A YTHDF2 reader is required for suppression of immune stimulation by circFOREIGN while the METTL3 writer is also needed for self/nonself-recognition. m6A UV-crosslinking and immunoprecipitation revealed that circSELF gained an m6A modification within 50–100 nt downstream from the circularization junction. When circFOREIGN was mutated for all m6A RRACH motifs, its RIG-1-mediated immunogenicity was increased by 10,000 fold.Other authors found another unexpected role of circRNA mresponse ,57. Theyachinery . The m6A6A machinery might be related to the presence of the exon junction complex (EJC) on this cirRNA spliced by endogenous spliceosome [6A reader YTHDC1. M6A modifications around the start codon prevent NMD. Knowing that induction of NMD is dependent on the presence of the EJC on spliced mRNA, we can speculate that an interaction between the EJC and METTL3 might explain the difference of methylation of circSELF and circFOREIGN. The concept of RNA marking as self or nonself constitutes an important parameter to take into account if using circRNAs as biotechnological vectors for protein expression.Association of circSELF with the miceosome . Supporticeosome . NMD-tarWhile there is no more doubt about translation of covalently closed circular RNA, the linearity of canonical mRNA can be questioned. It has been shown for more than thirty years that polysomes exhibit a circular or spiral form . This waRNA functional circularization is not limited to the interaction between eIF4G and PABP. It also takes place on nonpolyadenylated histone mRNAs via a 3′ terminal stem-loop that binds the SLBP protein. The 3′–5′ bridge is formed by the complex SLBP/MIF4G(SLIP1)/eIF3/eIF4F  Figure b. SeveraThe 3′ translational enhancers (3′TE) have been identified in several plant viruses such as the tobacco mosaic virus whose mRNA 3′UTR contains a pseudoknot that stimulates translation initiation in conjunction with the 5′cap . HoweverThe model of RNA looping was demonstrated in 2014 by Peak et al. who tethered eIF4G fused to MS2 coat protein to the 3′UTR of a reporter mRNA through MS2-binding sites . This fu6A modification [6A modifications in mRNA 3′UTRs, in particular in the vicinity of the termination codon [6A writer METTL3 and the eIF3 subunit h [6A, in addition to driving internal initiation of translation, participate in mechanism of mRNA circularization when located in the 3′UTR.An additional mechanism of mRNA closed-loop model involves the mfication . Me-RIP-on codon . Such moubunit h   are quite different . These aThe mechanism of ribosome recycling has been fully documented in the last decade, with several studies revealing a full connection between the steps of termination and initiation of translation. Termination is mediated by the eukaryotic release factors (eRF) 1 and 3 . eRF1 isThe concept of translation acceleration by ribosome recycling leads us back to translation of covalently closed circRNA, which may be subjected to the ribosome recycling process and are good candidates for the CLAR mechanism and the acceleration rate of translation initiation  vector expressing a circRNA coding GFP under the control of the CMV promoter, with the EMCV IRES to drive initiation of translation . Vector A recent study has developed a cell factory for recombinant protein production in Chinese hamster ovary (CHO) cells, based on rolling-circle translation . Cells wOur last word will be that the concept of RNA circle and of circular RNA translation can no longer be considered as an exception, and may be the key for efficient translation in eukaryotes. Translation of RNA circles also represents a powerful tool in future biotechnology and gene transfer research for the development of new gene therapies.


Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats ( In recent years, three coronaviruses (CoV) have emerged to have a significant impact on global health and the global economy. Two of these, which cause severe acute respiratory syndrome (SARS ) and MidAlphacoronavirus, and that the bats retain the virus for up to 4 months of hibernation. Since the bats we examined had been maintained as groups in isolated incubators or semi-isolated cages, we were able to test the hypothesis that little brown bats in the wild are infected with closely related variants of a coronavirus. We predicted that, since the bats were randomly assigned to different incubators/cages, (1) prevalence of infection should be similar across incubators and cages, and (2) any variation in viral genomes should be evenly distributed among cages and incubators.Access to a large number of archived samples from an unrelated experiment  gave us n=174, P value=0.817). A lack of blood samples prevented us from confirming viral prevalence by serological methods.To estimate the prevalence of coronavirus in little brown bats, and to determine the tropism of the virus, we performed PCR for the coronavirus RNA-dependent RNA polymerase (RdRp) gene on samples from the brain, intestines, liver, kidney and spleen of 157 little brown bats. We only detected the coronavirus in the intestines. On average, one-third of the bats  containeWe then compared the nucleotide sequences and the derived amino acid sequences of the PCR products. We included a corresponding segment from little brown bat coronavirus (KF430219) and from the Rocky Mountain coronavirus detected in a big brown bat (HQ336976) into the alignments. The sequences segregated into two distinct clades . The claMyotis lucifugus bat coronavirus (Myl-CoV) is 28 173 bases. We assigned open reading frames based on the published KF430219 sequence. The assembled sequence includes a 3′ poly-adenine tail, which is missing from the annotated KF430219 sequence.We assembled the entire genome of the coronavirus from RNAseq data from the intestines of seven bats (which contained clade 1 coronaviruses). The genome of the Alphacoronavirus. Myl-CoV is more closely related to Scotophilus bat coronavirus 512 and PEDV-CoV (CV777 strain). Other Alphacoronaviruses related to the Myl-CoV are human CoV 229E, NL63 and coronaviruses detected in other bat species   .Several coronaviruses have a predilection for respiratory as well as intestinal tissue. Although we had only detected Myl-CoV RNA in intestinal samples, we performed immunohistochemistry (IHC) to further explore the tropism of the virus. We were unable to detect coronavirus antigen in any of the tissues except for lungs. We detected the Myl-CoV antigen in the lung of five bats, all of which were positive for viral RNA in the intestines . In the We also performed transmission electron microscopy on portions of the lung sections that contained viral antigen . AlthougM. lucifugus were evaluated independently by two pathologists. Although the lesions were very mild, both pathologists had higher lesion scores for the categories of bronchiolar epithelial vacuolation and degeneration, bronchiolar epithelial hyperplasia and erosion of epithelium in virus-infected bats compared to uninfected bats. There was no obvious consistent inflammation of the bronchus and, although there was mild diffuse inflammation of the interstitium and alveoli, there was no difference between virus-infected and uninfected bats  is mainly present in the intestines and lungs; (b) co-hibernating little and big brown bats may share closely related coronaviruses; (c) Myl-CoV can persist in M. lucifugus for up to 4 months, the hibernation period of our experiment; and (d) the presence of Myl-CoV in bronchial epithelium is associated with minimal pathology or inflammation. However, a larger sample size under controlled conditions of exposure, with more rapid fixation of tissues, is required to fully characterize the lesions in lung and intestines.Although events of successful viral spillover to distantly related species are thought to be extremely rare, in recent years coronaviruses, including SARS-CoV , MERS-Coet al. [Dominguez et al.  and otheet al.  previouset al.  for the Complex strategies allow viruses to remain endemic in populations. These include: (1) a continuous source of susceptible hosts for viruses that cause short-term acute infections with long-lasting immunity (e.g. measles virus); (2) antigenic drift (e.g. influenza virus) of virus or waning immunity  that allows reinfection; and (3) long-lasting latent (e.g. herpesviruses) or persistent infections (e.g. pestiviruses) with sustained or periodic shedding. How bat viruses are maintained in their natural host populations, or how they avoid extinction as host populations become immune and less susceptible, however, is not understood. It is possible to establish persistent infections in cultured cells with viruses that may have originated in bats, including Ebola virus  and SARSBased on the sequence of the amplified portion on the RdRp gene, we observed considerable polymorphism among the Myl-CoV, with sequences segregating into distinct clades. Based on the maximum mutation rate possible for the RdRp gene , we shouAlphacoronaviruses. They concluded that these coronaviruses do not persist in their hosts, but are maintained in populations by the introduction of new susceptible individuals. Their results, however, do not rule out persistence in individual animals with low levels of virus replication and undetectable shedding, interspersed with short periods of increased replication and shedding. Our observations suggest that the Myl-CoV can persist in its hosts for at least the 4-month hibernation period. Due to strict biosecurity, spread of virus between incubators was unlikely and the distribution of variants among incubators (and cages) argues against spread within incubators (or cages). Our results therefore suggest that the bats were infected before they were collected.Osborne and others  were unaAlphacoronavirus, which includes three coronaviruses that infect human lungs and pig intestines . We confirmed the presence of virus in the intestine via PCR and in the lungs by immunohistochemistry, electron microscopy and PCR. However, we were unable to detect the Myl-CoV in the intestines using immunohistochemistry. The reason for this might be that the part of the intestine used for RNA extraction had the virus in it, whereas the part used for histology did not.Whole-genome phylogenetic analysis revealed that the Myl-CoV belongs to the genus M. lucifugus. It might also be that bat neutrophils are more efficient at controlling virus infection and obviating a massive neutrophilic infiltrate. An alternative explanation for the lack of inflammation in bats may be that cell necrosis occurs at the epithelial surface, with dead cells sloughing into the lumen. The cytokines required to stimulate inflammation and immunity might not enter the interstitium and the systemic circulation. An infection that is localized to epithelial cells without breaching the basement membrane is a good strategy for a virus to allow persistence of infection. However, in our study the sample size was small and it is difficult to reach a firm conclusion about the host’s response to coronavirus infection.Our histological lung sections provided a novel insight into the persistent infection of a coronavirus in its reservoir bat host. Firstly, we observed that the cells that were infected showed degenerative changes that resulted in rare multifocal areas of bronchiolar epithelial erosions, with no obvious inflammation targeting these lesions. The absence of neutrophilic infiltration is contrary to what has been seen in non-bat species affected by similar coronaviruses. Previous studies in transgenic mice showed that HCoV-229E infection led to massive neutrophilic infiltrate . Lung saAlthough our results demonstrate viral persistence during hibernation and do not address it in normothermic bats, we propose a model for the maintenance of the coronavirus in little brown bat populations . Bats arOur study demonstrates for the first time that several bats in a population can maintain a coronavirus infection through hibernation as an apparently non-pathogenic infection. Our observations also support growing evidence that natural and experimental viral infections in bats are not accompanied by acute inflammation and pathology.Pseudogymnoascus destructans, the causal agent of WNS. Male little brown bats were collected from two different WNS-free caves in Manitoba, Canada under the Manitoba wildlife scientific permits WB11145 and WB13148. Details for the segregation of bats into incubators and cages in the 2010–11 and 2011–12 experiments are in Bat tissues were obtained from two previous experiments  in the wBat tissues were homogenized and RNA was extracted using the RNeasy Plus mini kit , as per the manufacturer’s protocol. cDNA was prepared using the QuantiTect reverse transcriptase kit  as per the protocol mentioned in the kit.The sequences for all of the primers used in the PCRs are in Myotis lucifugus coronavirus (Myl-CoV) nucleocapsid gene (1278 bp) was amplified from intestines and lungs using a forward primer and a reverse primer  was sent to the Centre for Applied Genomics at the Hospital for Sick Children , where RNA quality was assessed using a Bioanalyzer (Agilent Technologies), Poly(A) mRNA was enriched using oligo dT-beads, and cDNA libraries were prepared using the NEBNext ultra directional RNA library prep lit (New England Biolabs). Barcoded libraries were pooled in equimolar quantities and sequenced on a HiSeq 2500 system (Illumina) to generate 126 bp paired-end reads.M. lucifugus genome sequence assembly (Myoluc 2.0) [in silico read normalization. We performed local blastn to search for sequence similarity between the Trinity-based transfrags and a coronavirus reference genome (KF430219). The sequence of the entire genome of the Myl-CoV was submitted to GenBank, accession number KY799179.Sequence data quality was assessed using FastQC v 0.11.5  and the luc 2.0)  in the sluc 2.0) , extractluc 2.0) , and pooEscherichia coli BL21-competent cells [−1 of lysozyme was added for 15 min to accentuate the process of bacterial cell wall breakdown. One ml of Halt protease and phosphate inhibitor cocktail  and EDTA was added and the blob of bacteria was sonicated for 60 s on ice. The supernatant was removed after centrifuging the bacteria at 15 000 r.p.m. in a Thermo F18 12×50 rotor (32 000 g) for 20 min and the pellet was treated with 1.5 % N-lauroylsarcosine (Sigma L-9150) to further disrupt the cells and then added onto the supernatant. From later SDS-PAGE analysis, we learned that the N-lauroylsarcosine treatment of the pellet led to the release of fusion protein from the cell. Fusion protein was purified from the supernatant using glutathione–Sepharose 4B beads , followed by elution using 10 mM glutathione for 16 h. Elution was performed twice to obtain protein concentrations of 6.1 and 4.3 mg ml−1. The protein was verified using SDS-PAGE .Myl-CoV N-pGEX-KG plasmid was transformed into nt cells . Five huPolyclonal Myl-CoV N anti-serum was generated in rabbits. This was carried out in strict compliance with the Canadian Council on Animal Care guidelines (protocol 20090050). Two rabbits were procured by the Animal Care Unit at the Western College of Veterinary Medicine. On days 1, 28, 42 and 56, nucleocapsid protein mixed with Titermax was injected into rabbits. Rabbits were bled to obtain serum 1 day prior to each antigen injection. Binding of antibody was verified using Western blot and immunofluorescence , after which we proceeded with immunohistochemistry.M. lucifugus tissues were obtained from our previous study [Eptesicus fuscus kidney (Efk) cells [Formalin-fixed paraffin-embedded (FFPE) blocks of us study  and sectk) cells  were useTissue sections for histopathology were stained with H and E by Prairie Diagnostic Services at the University of Saskatchewan. Each lung section was independently assessed for bronchiolar epithelial degeneration and inflammation by two veterinary pathologists, blinded to sample identity. Five categories of lesions were established: diffuse lung inflammation, inflammation of bronchus, bronchiolar epithelial vacuolation, bronchiolar epithelial hyperplasia and bronchiolar epithelial erosion. All lung sections from all bats were examined to determine the range of changes and then changes were scored between 0 and 3, with 0 indicating normal and 3 indicating the most severe change within the eight bats examined. The maximum score of 3 would still be considered mild within the typical pathology scoring system describing the severity of inflammatory response: mild, moderate or severe.Myl-CoV-infected cells in the bronchial epithelium were marked in the corresponding IHC slides, so that the specific location could be sampled for electron microscopy (EM). The slide was soaked in xylene to remove the coverslip, and soaked in xylene:propylene-oxide (PO) (2 : 1) and (1 : 1) for 30 min and 15 min, respectively, with this being followed by 1 h soaking in PO. PO : Epon (1 : 1) was consistently dropped on the slides for 1 to 2 h followed by pure Epon for 1 h. Labelled capsules were filled and inverted on the tissues on the slide. Polymerization was performed at 65 °C for 24 h. The block was broken off the slide, which lifted the section along with it, and was then sectioned on to 200 mesh copper grids, which were viewed by transmission electron microscope.mega7 [For the whole-genome phylogenetic tree, genome sequences of 33 representative coronaviruses and Myl-CoV were aligned using ClustalW (v 1.83) . Maximummega7 . DivergeFor the phylogenetic tree of the RdRp gene segment, The sequences obtained from Sanger sequencing were used, along with corresponding segments from KF430219 and HQ336976. Only sequences that were completely unambiguous were used in the analysis. The maximum-likelihood trees were constructed in a similar manner to that used for the entire-genome phylogenetic tree.Forty-one sequences of the RdRp gene, using Sanger sequencing, were submitted to GenBank (accession numbers KY820767 to KY820807). The Myl-CoV complete-genome sequence was submitted to GenBank (accession number KY799179). Nucleotide sequences for the phylogenetic tree were obtained from GenBank. The accession numbers of the sequences are AF353511 (PEDV.CV777), AF391541 (BCoV.ENT), AY585228 (HCoV.OC43.ATCC.VR.759), DQ001339 (Avian IBV.p65), DQ011855 (PHEV.VW572), DQ022305 (BtSARS.HKU3-1), DQ071615 (BtSARS.Rp3), DQ412042 (BtCoV.Rf1), DQ412043 (BtSARS.Rm1), DQ415904 (HCoV.HKU1.N6.GenotypeA), DQ445912 (HCoV.NL63.Amsterdam.496a), DQ648856 (BtCoV.273.2005), DQ648857 (BtCoV.279.2005), DQ811789 (TGEV), DQ848678 (FCOV), EF065505 (BtCoV.HKU4.1), EF065509 (BtCoV.HKU5.1), EF065513 (BtCoV.HKU9.1), EF065514 (BtCoV.HKU9.2), EF065515 (BtCoV.HKU9.3), EF065516 (BtCoV.HKU9.4), EF203067 (BtCoV.HKU.HK.33.2006), EU420137 (BtCoV.1B.AFCD307), EU420138 (BtCoV.1A.AFCD62), EU420139 (BtCoV.HKU8.AFCD77), FJ647225 (MurineCoV.MHV.A59), KC164505 (MERS-CoV.England1), NC002645 (HCoV.229E), NC004718 (SARS-CoV), NC009019 (BtCoV.HKU4.1), NC009657 (Scotophilus BtCoV.512.2005), NC009988 (BtCoV.HKU2) and KF430219 (Myotis CoV – USA.2006).2642Supplementary File 1Click here for additional data file.