RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation. Clinical samples from surgery and biopsy procedures are fixed for histopathological analysis. The aim of fixation is to preserve tissues permanently in as lifelike a state as possible and to make it possible to generate thin stained sections. In the past, an optimal morphological preservation was the sole requirement for fixation, whereas nowadays nucleic acids preservation for onco-pathology has also become an important requisite for gene expression profiling and sequencing aimed at defining reliable diagnostic and prognostic parameters . The 4% We here describe a novel method based on high performance liquid chromatography (HPLC) that is aimed at investigating on the amount of unmodified nitrogenous bases in RNA extracts obtained from different procedures of fixation.To set-up the HPLC-based method to detect unmodified nucleotide monophosphates (NMPs) in RNA extracts, we first set-up the experimental conditions to quantify each unmodified NMP by HPLC and subsequently we investigated methods to degrade RNA into NMPs. The study design is divided into three main sections, as described in 1H NMR and by HPLC  to the nitrogenous bases [Formalin fixation modifies nucleic acids by the addition of a mono-methylol group  in 1× digestion buffer was injected into HPLC to identify peaks related to non-template control as shown in ®, with and without paraffin embedding in comparison to untreated samples (frozen specimen) following ISO standard for pre-analytical processes [The aim of the present study was to apply the HPLC method to fixed samples. To this end, mouse livers were used for the analysis set-up. Mouse livers were submitted to different fixation procedures for 24 h, namely neutral buffered formalin, Bouin’s solution and RCL2rocesses .Each HPLC analysis was carried out in triplicate loading 600–700 ng of digested RNA in each injection. Chromatograms, where peaks related to the blank were subtracted, are shown in Regarding hm-NMPs, the chromatograms in RNA purity was measured through the A260/280 and A260/230 ratios by the NanoDrop™ ND-1000. The mean A260/280 ratio was 1.98 (range 1.70–2.10), reaching the lower value in BF liver specimen. A260/230 ratio was similar in all samples with an average of 2.11 (range 2.07–2.14) as reported in Integrity of RNA extracts was investigated by the Agilent 2100 Bioanalyzer  recording the RIN number and quantifying the different RNA fragments see . As alreThe relative amount of RNA stretches was retrieved by the fragment size analysis using the Agilent 2100 Bioanalyzer. Stretches of 60–149 nt prevailed in Bouin’s fixed mouse livers, in spite of the embedding process. On the contrary, for formalin and RCL2 a comparable amount of 60–149 nt and 150–299 nt fragments were observed, as shown in In addition, the RNA integrity was investigated in mouse livers tissue by amplifying different stretches of the cytochrome C oxidase I, mitochondrial (mt-CO1) gene (NC_005089.1) , charactp < 0.0001, p = 0.03, p = 0.04 and p = 0.01, respectively). The FFPE and RFPE slopes were closer to the slope of the fresh frozen sample , 10 FFPE tissues of different cancer types and three RNAs obtained from peripheral blood. All the samples showed a reduced amount of the canonical NMP, down to 31% in a 28-years old cervical uterine cancer. However, no relationship was found between the aging of the samples and the percentage of the canonical NMPs.After the de-modification step , only hm-GMP was detected in the chromatogram with different extent in all FFPE samples as well in BFPE samples. The ratio among peak areas (hm-GMP vs. GMP) was 0.02 in Bouin’s sample 1, 0.03 in Bouin’s sample 2, an up 0.28 in FFPE tissues. Furthermore, peaks at higher retention times (≥40 min) were recorded in all the samples, with major amplitude in FFPE samples likely related to dimers (methylene di-adducts) between nitrogenous bases as resulting from the cross-linking process.The integrity of RNA extracts was investigated by the Agilent 2100 Bioanalyzer  recording the RIN number and quantifying the different RNA fragments. Gel-like images of the BioAnalayzer runs are available in the In all fixed samples, the most representative fractions were related to fragments of 60–299 nucleotides as reported in Tissue fixation is a compulsory step for preserving cellular and tissue morphology for pathological examination. Fixation by itself introduces artifacts, involving some chemical modification of tissue components used to prevent their loss during tissue processing . With reper se does not reveal the quality of RNA molecules in terms of fragment lengths, but rather it allows investigating the chemical integrity of each of the four canonical NMP of the RNA. The method in fact allows both identification and quantification of the NMPs released by the enzymatic digestion with nuclease S1 from as low as 600–700 ng of RNA. Consequently, in agreement with our expectation, only the four canonical NMPs were detected in our control samples. In addition, although non-quantifiable owing to the unavailability of commercial standards, home-synthesized hydroxymethyl NMPs  were efficiently resolved as well.The method Out of the hm-NMPs, only hm-GMP was detected in our series of FFPE mouse liver samples as well as in clinical samples. Among hydroxymethyl nucleotide monophosphates, hm-AMP was reported to account around 40% of Adenines , but it In our series of mouse livers, differences were detected in the HPLC profile of formalin-fixed samples when compared to FFPE ones. Higher values of canonical NMPs were assessed in the former (85% vs. 80%) and no peak referring to hm-GMP was detected as well. The latter result could have different explanations. From one side it is possible that the absence of the dehydration process in fixed sample and the subsequent treatment of the sample in aqueous phase for the RNA isolation allows the re-conversion of hm-GMP to GMP, as observed for hm-dAMP . It is aBoth formalin and Bouin’s solution are formaldehyde-based fixative, but different patterns of modification were found by HPLC analysis in mouse livers. The amount of unmodified NMP was always higher in Bouin’s fixed samples when compared to formalin-fixed ones (96–100% vs. 80–85%) Bouin’s fixative is made up of picric and acetic acids as coagulant components of the fixative and formaldehyde as cross-linking one . RNA fro® proved to preserve better RNA after fixation assuring about 99% of unmodified NMPs, while this value lowered to 88% after embedding procedure, likely due to the procedure itself [In our series of mouse livers, the quality of samples in terms of RT-PCR seems to be higher in fixed and paraffin-embedded specimens if compared to fixed ones, independently of the fixative used. That apparent discrepancy can find a possible explanation in fixed sample treatment, which were submitted to fixation, washed and frozen at −80 °C. For RNA isolation, they were thawed and submitted to mechanical homogenization. The fact that fixed samples were not dehydrated, maintaining residual water in tissues and that a freezing/thawing cycle was applied together with the heating due to mechanical homogenization might account for that difference. This is further supported by the intrinsic properties of mt-CO1 gene , which has been reported to be resistant to RNAse degradation, but heat-sensitive . That mae itself . By compThe HPLC method was also applied to a set of aged clinical samples see , which s®, as possibly most alcohol-based fixatives, does not induce chemical modification of the nucleotide monophosphates. In addition, our data support that Bouin’s solution prevents, in ISO standard pre-fixation conditions, the cross-linking activity of formaldehyde. Furthermore, we have shown that hm-GMP is the most represented hm-NMP. We acknowledge as a limitation of this method that it allows the quantification on unmodified NMPs as indirect proof on the non-modification of RNAs, without any additional information on the degradation of the transcripts, which is mandatory for further molecular analysis. Nevertheless, the presented method could easily be applied for analyzing the effect of novel fixatives and/or RNA de-modification procedures. Consequently, these data on the molecular composition of the template, coupled to conventional and newly proposed methods, such as the PERM and DV algorithm from BioAnalyzer for assessing the molecular weight of the templates, are helpful in the evaluation of RT-qPCR- based results that can be achieved from archived fixed and paraffin-embedded samples. Even in long-term stored FFPE tissues, only hm-GMP and di-adducts were found after the de-modification step of the RNA, without correlation with the age of the blocks. The present results stress that pre-analytical procedures play a major role in the quality of the clinical specimens, thus confirming that the use of the ISO standard document [In conclusion, the HPLC method here described makes it possible to detect the four canonical NMPs of the RNA as well as their hydroxymethyl derivatives. Most importantly, when applied to less than 1 µg of RNA from fixed tissues, it provides valuable data on chemical modification of RNA molecule as produced by fixation processes. It has been possible to demonstrate, indeed, that RCL2document  guaranteTM Ribunclease . For assessing the digestion conditions, 5 µg of each 15 bases oligo  as well 5 µg of a control RNA purchased by Celbio were incubated in 1× working buffer with 50–160 units of enzyme at 37 °C for two hours in a final volume of 50 µL. After digestion, the samples were immediately stored at −20 °C up to HPLC analysis. The following digestion reactions of mouse liver as well as clinical samples were carried out using about 2 µg of RNA with 80 units of S1 Nuclease  in the abovementioned conditions.Hydrolysis of the nucleic acids was achieved by enzymatic reactions which cleave the RNA molecule to 5′-mono-nucleotide. Three different enzymes were tested: S1 Nuclease , S1 Nuclease  and RNAase ONEFour wild type Black Swiss mouse livers were collected at the ICGEB animal house after sacrifice. Each liver was washed in PBS, blotted onto a paper and immediately chilled on wet ice for transport. At the pathology department, livers were subdivided into fragments and submitted to fixation. Each liver tissue was equally represented in the different treatments. According to ISO standard  a duplic4)2HPO4 + 0.76 g (NH4)H2PO4 + 2.6 mL of 40% TBAH solution were mixed in water. The phosphate buffer was filtered through a 500 mL Nalgene™ Rapid-Flow™ Sterile Disposable Filter Unit with PES 0.2 µM membrane prior to use. To quantify unmodified nucleotides monophosphate 1a–4a (2) of 0.999. Fixed samples were spiked with each nucleotide-monophosphate-disodium salt to definitely identify the unmodified 1a–4a nucleotide peaks in HPLC analysis. Fixed samples were diluted 1:10 in phosphate buffer and injected in HPLC in triplicate analyses for a run time of 60–70 min.HPLC analyses were run on a non-thermostated Agilent series 1100 liquid chromatograph equipped with a Kinetex C18 250 × 4.6 mm 5 µm 100 Å  column with a column guard, a 20 µL loop and a UV detector at 260 nm. The flow was set to 1 mL/min. The eluent was phosphate buffer 0.03M (pH 7.4) + tetrabutylammonium hydroxide (TBAH) 10 mM. For 1 L phosphate buffer in water (mQ): 3.53 g  using a homogenizer . The homogenization was carried out in wet ice for 20 s for four times at 20,000 rpm. Successively, 200 µL of Lysis solution and 15 µL of proteinase K were added to the homogenized samples and processed following the instructions of Maxwell® RSC RNA FFPE kit (Cat. No. AS1440). Total RNA was automatically eluted in 50 µL of Nuclease-free water, split into aliquots and stored at −80 °C. RNA concentration and purity were measured by absorbance through NanoDropTM ND-1000 spectrophotometer .On average 35 mg of formalin, Bouin and RCL2 fixed tissues were cut on a cold surface and transferred into a cleaned 2 mL vial. The homogenization was performed with 50 µl of Lysis solution obtained by the MaxwellRNA was isolated from fixed and paraffin-embedded mouse liver samples and from routine clinical tissues. With regards of clinical tissues, two Bouin’s fixed and paraffin-embedded high-grade ovarian cancer samples and ten cancer FFPE samples of different origin were used. Those specimens were collected at the National Cancer Institute of Aviano and at the University Hospital of Trieste. Informed consent was obtained from participants included in the study and ethical approval was obtained by the Institutional Review Board of CRO-Aviano  and by the ethical committee of the University of Trieste .® RSC instrument  following the instruction of the Maxwell® RSC RNA FFPE kit (Cat. No. AS1440). The protocol of isolation includes a digestion step at 56 °C and a de-modification step of 1 h at 80 °C. After RNA isolation, samples were split into aliquots and stored at −80 °C. RNA quantification and purity detection were assessed by NanoDropTM ND-1000 spectrophotometer .RNA was isolated from four 10 µm-thick sections of fixed and paraffin-embedded specimens using the MaxwellTM ND-1000 spectrophotometer . Afterwards, an aliquot of RNA was submitted to DNAse digestion.RNA was isolated from peripheral blood from healthy volunteers by the use of Tri-reagent . Samples were split into aliquots and stored at −80 °C. RNA quantification and purity detection were assessed by NanoDropThirty µL of total RNA were treated with 20 U of DNase I  and 20 U of RNase Inhibitor  at 25 °C for 20 min. Then the reaction was stopped with 4 µL of EDTA 25 mM at 65 °C for 10 min as already described . SamplesRNA integrity was assessed through automated electrophoresis by the Agilent 2100 Bioanalyzer instrument . One microliter of each sample was loaded into the sample well of the Agilent RNA 6000 Nano Chip  which was previously filled with the gel-dye mixture and the 5 µL of RNA 6000 Nano Marker, as suggested by the manufacture’s instruction . In addition, the distribution of different length fragments was measured by the relative abundance in comparison to the total RNA area. The five ranges were defined as follows: from 1 to 59 nucleotides (1), from 60 to 149 (2), from 150 to 299 (3), from 300 to 449 (4) and equal or over 450 (5).2 and 8 U RNase Inhibitors  as already reported [® ep Realplex  using the following cycling conditions: 95 °C for 2 min, 45 cycles of 95 °C for 20 s, 30 s of annealing temperature and the extension at 72 °C for 30 or 45 s (extension time of 45 s was used for 302 bases amplicon). Annealing temperature was 60 °C for 60 bases amplicon, 57.5 °C for 179 bases amplicon and 61 °C for 302 bases amplicon. At the end of the protocol, the melting curve analysis was carried out to evaluate the specificity of the amplified products.For mt-CO1gene amplification 200 ng of mouse RNA were reverse transcribed using 250 U M-MLV reverse transcriptase , 1 mM dNTPs, 0.01 M DTT, 4.5 mM MgClreported . The ampreported . For eacThe ∆Amp method  was applLinear regression was performed by plotting the Ct values versus the amplicon size for each pre-analytical condition. ANCOVA test was run to investigate on regression lines, in detail to test if slopes and intercepts were significantly different. Bonferroni analysis was carried out to inspect multiple comparison among all samples. All the statistical analyses were carried out using the GraphPad Prism 8.0 software .


Major 5′terminally deleted (5′TD) group-B enterovirus (EV-B) populations were identified in heart biopsies of patients with fulminant myocarditis or dilated cardiomyopathy suggesting that these 5′TD forms are key drivers of host-cell interaction in EV cardiac infections. To date, early emergence of EV-B 5′TD forms and its impact on type 1 IFN response during acute myocarditis remains unknown. Using quantitative RACE-PCR assay, we identified major EV-B 5′TD RNA populations in plasma or heart samples of acute myocarditis cases. Deletions identified within the 5′ non-coding region of EV-B populations only affected secondary-structural elements of genomic RNA domain I and were distinguished in two major groups based on the extent of RNA structural deletions. Proportions of these two respective EV-B 5′TD population groups were positively or negatively correlated with IFN-β levels in plasma samples of myocarditis patients. Transfection of synthetic CVB3/28 RNAs harboring various 5′terminal full-length or deleted sequences into human cultured cardiomyocytes demonstrated that viral genomic RNA domain I possessed essential immunomodulatory secondary-structural elements responsible for IFN-β pathway induction. Overall, our results highlight the early emergence of major EVB-TD populations which deletions affecting secondary–structures of RNA domain I can modulate innate immune sensing mechanisms in cardiomyocytes of patients with acute myocarditis. Picornaviridae family and are recognized as major causes of aseptic meningitis, upper or lower respiratory tract infections and acute myocarditis cases diagnosed in neonates, infants and young adults2. Between 10–20% of acute myocarditis cases will evolve into chronic myocarditis as well as dilated cardiomyopathy 1.Group-B Enteroviruses (EV-B) belong to the 3. A study in 2008 of heart tissue from a patient who died of fulminant myocarditis demonstrated the presence of a group B Coxsackie virus type 2 (CVB-2) population with partial deletions at the 5′ terminus and proceeding inward4. Similar findings had been described following CVB-3 replication in cell cultures and in mice6. Bouin et al.7. These results were subsequently extended by NGS (Next Generation Sequencing) investigation of explanted heart tissues from a cohort of unexplained DCM adult patients8. Together, these findings suggested that EV-B populations with 5′ terminally deleted (5′TD) genomes can be generated and selected in heart tissues during the early acute viral myocarditis. The emergence of these low replicative TD populations offer a valid explanation on how EV-B can persist in hearts long after the acute infection during the chronic myocarditis of the clinical phase of DCM.EV-B RNA genome is approximately 7,400 nucleotides (nt) in length and is flanked at the 5′ end by a highly conserved non-coding region (5′NCR) that is crucial for the initiation of the viral replication and translation activities11 altering formation or stability of viral replication complexes and consequently decreasing genomic replicative capacities of 5′TD viral RNA forms14. Moreover, it was evidenced that the 5′triphosphorylated cloverleaf structure of CVB3 RNA is sufficient to induce a type 1 IFN response by the RIG-I signalling pathway10. In addition, it was reported that complete double-strand RNA replicative forms of EV-B were recognized by MDA5 triggering a significant type 1 IFN response in cultured human or mice cells9. Early natural deletions into the 5′NCR of EV-B populations could modify the RNA secondary-structural elements recognized by cytoplasmic immune sensors, such as RIG-I or MDA5 , resulting in a modulation of type 1 IFN pathway induction in human cells, a potential way to overcome antiviral immune innate host response leading to persistent infection in target tissues15. To date, the impact of 5′TD viral RNA forms on viral replication activities and type 1 IFN response levels remain to be fully investigated in EV-B induced acute myocarditis.Natural deletions within the 5′NCR of EV-B RNA populations can affect functional secondary-structural elements of the RNA domain I In the present report, using a quantitative RACE-PCR system we identified major EV-B 5′TD RNA populations associated with minor full-length (FL) viruses in peripheral blood or heart tissue samples of acute myocarditis pediatric patients. Identified deletion groups within the 5′NCR of major EV-B RNA populations affected only secondary-structural elements of the RNA domain I (cloverleaf). Using correlation analyses, we assessed the impact of EV-B 5′TD population proportions on viral genomic replication activities and type 1 IFN  secretion levels in peripheral blood samples of myocarditis patients. Subsequently, transfection of synthetic CVB3/28 RNAs harboring various 5′terminal full-length or deleted sequences were generated in vitro and their impact on type 1 IFN pathway induction was assessed on HeLa229 and human primary cardiomyocytes (HCM) cultures.6 copies [range 3.97 × 104–9.65 × 106] per microgram of total extracted nucleic acids ] median time delay between symptoms and sample collection was 3.58 days (1—7 days) (Table 1). An EV-genus specific real-time quantitative reverse transcription PCR protocol demonstrated an endomyocardial standardized median viral genome load value of 4.50 × 107. Our RACE-PCR assay allowed to size viral cDNA with 5′deletions ranging from 35 to 137 bp with an accuracy of 10% established by manufacturer, even in cases of mixed RNA forms   PCR followed by micro-electrophoresis assay 2 = 0.999, P < 10–3)  in length which was associated with minor proportions of 5′TD 15–36 nt (3%) and FL RNA forms (7%)      Fig. C. Intere05) Fig. A,B. By c07) Fig. D. Moreov2 = 0.880; P = 0.006)    Fig. C. By conMajor 5′terminally deleted (5′TD) Group-B enterovirus (EV-B) populations were previously identified in heart biopsies of patients with fulminant myocarditis or dilated cardiomyopathy suggesting that these 5′TD forms are key drivers of host-cell interaction in EV cardiac infections. In the present report, two major 5′TD RNA population groups were identified in peripheral blood or heart tissue samples of EV-B acute myocarditis cases and their respective proportions appeared to be positively or negatively correlated with type 1 IFN levels. To confirm immunomodulatory effects of these 5′TD RNA forms on the type 1 IFN pathway induction, synthetic CVB3/28 RNAs harboring various 5′terminal full-length (FL) or deleted genomic sequences were transfected into HeLa229 or human primary cardiomyocytes cultures. This in vitro approach displayed that EV-B genomic RNA domain I (Cloverleaf (CL)) possessed essential immunomodulatory secondary-structural elements responsible for IFN-β pathway induction in human target cells.16 and in a recent cohort of EV-induced DCM  binding site remains intact  particles21. Further experiments based on co-transfection of synthetic CVB3/28 FL and various 5′TD RNA forms in human cultured cardiac cells are needed to explore this hypothesis.Interestingly, taking into account the secondary structure of the EV-B RNA cloverleaf (RNA domain I), 5′TD viral populations were distinguished as two major groups (8–36 nt and 37–50 nt EV-B 5′TD forms) based on the extent of RNA deletions Fig. . Such te17. Interestingly, in only one study subject, the distribution of median levels of 5′TD viral forms appeared not to be significantly different between peripheral blood and heart compartments in an intra-patient comparative analysis, suggesting that peripheral blood circulating viral RNA population levels detected in the plasma blood were representative of those detected in cardiac tissues . It is intriguing to consider that the quantitative detection of various minor EV-B RNA populations could be a potential peripheral blood biomarker of acute myocarditis severity  transcription and translation levels in infected cells, a potential way to overcome antiviral immune innate host response.We showed for the first time that 8–36 nt and 37–50 nt EV-B 5′TD population’s proportions could modulate IFN-β levels in peripheral blood samples of acute myocarditis patients. We observed that these two groups of 5′terminally RNA deletions differentially affected secondary–structures of RNA domain I which could result in a differential qualitative binding of EV-B 5′TD RNA sequences to the RLRs (RIG-I or MDA5) acting as major innate immune sensors during EV-B acute infectionsIn summary, our results highlight the early emergence of major EV-B 5′TD populations which deletions affecting secondary–structures of RNA domain I can modulate innate immune sensing mechanisms in cardiomyocytes of patients with acute myocarditis. These results provide new insights into the role of EV-B 5′TD RNA forms on innate immune mechanisms and inflammatory responses in heart tissue and should stimulate the research for the development of new therapeutic strategies based on modulation of type 1 IFN pathway in EV-B induced acute cardiac infections.23; (ii) they were positive for EV RNA genome detection; (iii) they were negative for the molecular detection of HHV1-HHV6, PVB19 and adenovirus genomes and for the detection of a bacterial sepsis.Peripheral blood plasma (n = 6) and cardiac tissue samples (n = 4) were collected between 2014 and 2017 and stored in three biobanks (−80 °C)  were selected because: (i) they were collected from patients demonstrating a final recorded clinical diagnosis of acute myocarditis established according to ESC guidelines by physicians from the French myocarditis study groupThe institutional review committee  approved the study, and informed consent was obtained from the patients or subjects’ families at the time of hospitalization or necropsy. Our investigations conformed to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.HeLa229 cells were grown in minimum essential media (MEM) supplemented by 1% Penicillin–streptomycin (PS) , 1% L-Glutamine and 10% Fetal Bovin Serum (FBS) . Human primary CardioMyocytes  were cultured in cardiac myocyte medium (CMM) supplemented by 1% Penicillin–streptomycin, 1% Cardiac Myocyte Growth Supplement (CMGS) and 5% FBS (ScienCell Research Laboratories) at 37 °C with 5% CO2. Cardiac myocyte cell cultures were passaged twice a week and the cells were cultured between passages 3 to 9.Reverse transcription was carried out using Superscript II reverse transcriptase . Detection of full-length RNA copy number in heart tissues and peripheral blood samples was quantitated by RT-qPCR using a StepOne plus real time PCR system (ThermoFisher Scientific). Detection of viral RNA was performed using iQ Supermix (BioRad) and 200 nM NC1M primer (nt 456–474) (Fwd: 5′-CCCTGAATGCGGCTAATCC), 200 nM E2 primer (nt 582–601) (Rev: 5′- ATTGTCACCATAAGCAGCCA) and 100 nM S-Ent probe (nt 539–566) (Fwd: 5′- FAM-AACCGACTACTTTGGGTGTCCGTGTTTC-BHQ1) (Supplementary Table S1). RT products were unhybrized at 94 °C for 5 min, and then cDNAs were amplified during 45 cycles as follows: 94 °C for 15 sec, 63 °C for 1 min, and 68 °C for 30 sec. To validate detection and quantitation of EV sequences in clinical samples, serial dilutions of transcripts of wild-type CVB3 (CVB3/28 strain) clones were used as standard for qPCR.www.clustal.org/). Evolutionary distances were calculated using the Kimura 2-parameter method. Trees were constructed using the mixed-linear method (MLM) as implemented in MEGA 6 software24. Bootstrap values from 1,000 replicates are shown at the nodes. Scale bar indicates number of nucleotide substitutions per site.Molecular phylogeny of Enterovirus strains detected in clinical samples was based on the VP1 region nucleotide sequence. Sequences were aligned using Clustal W version 1.81  using QIAamp Viral RNA mini kit (Qiagen). cDNAs were generated using Superscript II kit  with random hexamers primers  in a 20μL reaction mix according the manufacturer’s protocol. cDNAs were then amplified by a two-step semi-nested PCR. Briefly, reverse transcribed products were amplified by KAPA Taq polymerase kit consisting of 1X KAPA TaqA buffer, 200 μM dNTPs, 400 nM of forward primer 222 (Fwd: 5′-CICCIGCIGGIAYRWACAT), 400 nM of reverse primer 224 (Rev: 5′-GCIATGYTIGGIACICAYRT) for the first PCR and 400 nM AN88 (Fwd: 5′-TACTGGACCACCTGGNGGNAYRWACAT), 400 nM AN89 for the second PCR (Rev: 5′- CCAGCACTGACAGCAGYNGARAYNGG), 1U KAPA Taq polymerase in a mix of 25μL with a cycling process composed of one cycle for 1 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 55 °C, 2 min at 72 °C, finishing in a cycle of 2 min at 72 °C. Amplicons were analyzed on Ethidium-bromide stained 1.5% agarose gels in TBE Buffer  and DNA bands were gel-purified using QIAquick Gel Extraction Kit (Qiagen) according the manufacturer’s instructions. Purified PCR products were sequenced by Genewiz  to detect VP1 gene using AN232 forward primer (5′-CCAGCACTGACAGCA) at 2,602 to 2,616 nucleotide position and AN233 reverse primer (5′- TACTGGACCACCTGG) at 2,977 to 2,963 nucleotide position7 and was incubated 5 min at 65 °C and then 5 min on ice. The 5′extremity of cDNA was then ligated with 1X T4 DNA ligase buffer , 50 nM Trp1 DNA adaptor for 5′NCR terminal deletion identification (Fwd: 5′- H-CCTCTCTATGGGCAGTCGGTGAT), 5 units of Ambion T4 DNA Ligase  was added to the cDNA-mix and was incubated overnight at 16 °C. Positive cDNA were amplified by a classical PCR reaction using KAPA Taq polymerase kit consisting of 1X KAPA TaqA buffer, 200 μM dNTPs, 400 nM AvCRev, 400 nM Trp1 (Fwd: 5′-CCTCTCTATGGGCAGTCGGTGAT), 1U KAPA Taq polymerase in a mix of 25μL with a cycling process composed of one cycle for 3 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 55 °C, 30 s at 72 °C, finishing in a cycle of 30 s at 72 °C. A 2% agarose gel electrophoresis was performed in order to control the length of the PCR products. Amplicons were then sized and quantified by Bioanalyzer High Sensitivity DNA Analyses using Agilent High Sensitivity DNA Kit (Agilent) according to the manufacturer’s instructions. The area of each relevant electrophoresis peak were integrated using software integrated analytic approach 28. The quantification ranges of the RACE-PCR assay followed by a micro-electrophoresis device were assessed using serial tenfold dilutions of synthetic full-length or 5′terminally deleted CVB3 forms in human cardiac or plasmatic total RNA extracts of EV-negative human samples.Total RNA was extracted from heart tissues and peripheral blood samples taken from patients with acute myocarditis using QIAamp Viral RNA mini kit (Qiagen). Viral RNA (200 ng) were reverse transcribed using Superscript II kit  with 400 nM AvCRev (Rev. :5′-AACAGGCGCACAAAGCTACCG)5 HCM or HeLa229 cells per well were seeded in 24-well plates . The plates were incubated overnight at 37 °C. Cells were washed with DPBS (Thermo Scientific) was added. The transfection mixtures (0.1 mL) consisted of 1 µg of synthetic RNA and 1 μl of Lipofectamine 2000 (Thermo Scientific) in Opti-MEM Glutamax. The mixtures were incubated for 20 min at room temperature. Then 100 μl of the mixture was added to the cells, and plates were transferred to a cell incubator at 37 °C for 8 h. The supernatants were collected, and the cells lysed by 500µL of TRI Reagent .2.0 × 10RNA was extracted from cultured cells with TRI Reagent  and reverse transcribed using SuperScript II Reverse Transcriptase (RT) (Invitrogen Life Technologies) following to the manufacturer’s instructions. cDNA was subjected to PCR using PowerUp SYBR Green Master Mix (2X) (ThermoFisher Scientific) to detect IFN-β expression. The primers used for PCR detection as follows: for human IFN-β, forward primer AGCTGAAGCAGTTCCAGAAG and reverse primer AGTCTCATTCCAGCCAGTGC. Primers specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward primer AGGGCTGCTTTTAACTCTGGT and reverse primer CCCCACTTGATTTTGGAGGGA) was served as the internal control. PCR was carried out in StepOnePlus Real-Time PCR Systems (ThermoFisher Scientific) programmed as follows: 94 °C for 15 s, 63 °C for 10 s, and 72 °C for 15 s for a total of 40 cycles, before melting curve. Results were analyzed by the ΔΔCT method, where CT is threshold cycle, and normalized to GAPDH mRNA. Data are represented as levels of mRNA relative to the mock transfected control samples and are displayed as the means ± SD of results from at least three independent experiments.For the quantitative determination of human IFN-β concentrations in supernatants of cultured cells, ELISA assays using commercially available Human IFN-beta DuoSet ELISA kits  were performed according to the manufacturer’s instructions.The dephosphorylation of synthetics RNAs was carried out using Antarctic phosphatase (10 U), 1X Antarctic Phosphatase buffer  and 2 µg of synthetics RNAs in 20 µL final volume for 1 h at 37 °C. Enzymes were deactivated at 80 °C for 2 min. RNAs quality was controlled by loading 8 µL of each controls and dephosphorylation products by electrophoresis on 1% agarose gel.All statistical analyses were performed using GraphPad Prism 7 (GraphPad) and SAS version 9.4 (SAS Institute Inc.). Quantitative variables were compared using the Mann Whitney U test and a P-value < 0.05 was considered as statistically significant. Linear regression was performed, and slopes were compared using Spearman correlation test. Because selected human biological samples  were of very limited availability and because number was not extendable; we did not have prior data to calculate it and we did not a priori calculate the number of subjects to include in the present study.We declare that all the data supporting the findings of this study are available within the paper and the Supplementary Information files.The study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedure, because the human biological samples (blood and cardiac tissue samples) as well as RNA extracted from HeLa229 and human primary cardiomyocytes used in our experiments remain limited biological sources.Supplementary file1


SARS-CoV-2 is still rampaging throughout the world while the many evolutionary studies on it are simultaneously springing up. Researchers have simply utilized the public RNA-seq data to find out the so-called SNPs in the virus genome. The evolutionary analyses were largely based on these mutations. Here, we claim that we reliably detected A-to-G RNA modifications in the RNA-seq data of SARS-CoV-2 with high signal to noise ratios, presumably caused by the host’s deamination enzymes. Intriguingly, since SARS-CoV-2 is an RNA virus, it is technically impossible to distinguish SNPs and RNA modifications from the RNA-seq data alone without solid evidence, making it difficult to tell the evolutionary patterns behind the mutation spectrum. Researchers should clarify their biological significance before they automatically regard the mutations as SNPs or RNA modifications. This is not a problem for DNA organisms but should be seriously considered when we are investigating the RNA viruses. The outbreak of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) in the beginning of year 2020 has caused severe damage to China especially the Hubei province –3. RecenPapers on the evolutionary patterns of SARS-CoV-2 have emerged as rapidly as the outbreak of virus. Several studies downloaded the publically available RNA-seq data and performed “SNP calling” or sequence alignment. The SNP distribution or frequency spectrum acts as a super informative inference of selection patterns. The recent study has discussed topics on the origin and continuing evolution of SARS-CoV-2. Although the methodology of some studies was challenged by other researchers , their nTo better illustrate the pipeline of SNP calling, let us take the human genome for instance. The SNP calling process is accomplished by mapping the DNA-seq reads (of a sample) to the human reference genome, and the reliably detected mismatch sites should be SNPs. If the RNA-seq data of the same sample is available, one could find the same nucleotide changes from the reference genome to the RNA-seq reads, indicating that the mutation takes place at DNA level. However, one could not determine the direction of the mutations without an outgroup species. In contrast, if a variation site is only found in the RNA-seq reads but not the DNA-seq reads, then this is possibly an RNA modification site. For example, the vertebrate adenosine deaminase would change adenosines to inosines , which wResearchers should note that SARS-CoV-2 is a positive strand RNA virus. The so-called reference genome is actually the RNA sequence. Without a DNA template, the mismatches found between the reference and the RNA-seq reads could either be a “SNP” or an RNA modification site. It is futile to try any filtering cutoffs on these variation sites because the SNPs and RNA modification sites are technically indistinguishable. Application of cutoffs only makes the variants more reliable but does not help distinguish whether the variants are SNPs or RNA modifications. Even when multiple outgroup species are available, the reference sequence (RNA) of the outgroup viruses may also undergo the same RNA modification process (by host cells), making it difficult to define the ancestral state and the direction of mutations.Fig 1 and In this study, using a well-established mutation finding pipeline (see https://www.ncbi.nlm.nih.gov/genome/). The coding sequences were extracted according to the genome annotation. The RNA-seq data were retrieved from NCBI under accession numbers SRR10903401, SRR10903402, SRR11059942, and SRR11059945.We downloaded the novel coronavirus SARS-CoV-2 genome from the NCBI website . Reads with too many mismatches could not be aligned to the reference genome. However, the multiple clustered mismatches could be RNA modification events. To retrieve more RNA modification sites in clusters like this, we manually transformed the reference sequence and the RNA-seq reads [We mapped the RNA-seq reads to the CDS of SARS-CoV-2 using BWA mem  with defeq reads . We perfeq reads , 9. Howeeq reads .Codes of mapping and variant calling:Mapping:bwa index reference.fastabwa mem reference.fasta sample.fastq > sample.samVariant calling:samtools mpileup–q 25 –Q 35 sampleX.sam > sampleX.vcfWe used the R language to perform the statistical analyses and graphic work. We also used EXCEL to plot some figures when necessary.Fig 1) and look at the clustered modification events across the transcriptome.A recent study  dealt wiFig 2A). There are 5310 (59.1%) A-to-G mismatches and 2015 (22.4%) G-to-A mismatches, and the ten other types of mismatches composed only 18.5% of the totally 8989 mismatches . The most prevalent A-to-G mismatches could be interpreted as the adenosine-to-inosine deamination conferred by the host cells. For the second-most prevalent G-to-A mismatches, it is possible that the reference sequence (RNA) of SARS-CoV-2 itself suffered from A-to-G deamination by the host cells, and the nucleotide in the RNA-seq is the unmodified version (adenosine). The third-most abundant T-to-C and C-to-T mismatches might represent the cytosine-to-uridine deamination system in the host cells.We downloaded the reference and a set of RNA-seq data of SARS-CoV-2. We mapped the RNA-seq reads to the reference sequence with a well-established pipeline . To technically validate the reliability of the mismatch sites, we manually extracted a 150bp read and aligned it to the reference sequence . The A-to-G alterations are clearly presented in the alignment. Note that the terminology “error” could refer to mis-alignments or sequencing errors. The validation here is to check the accuracy of the mapping pipeline. The manually examined read only proved that the sequence alignment is reliable. The control for sequencing errors would be discussed in the following section.We treated the A-to-G as the A-to-G RNA modification sites in the virus sequences. We found that the density of A-to-G modification varied moderately across different genes . There are 2878 (62.5%) unique A-to-G variation sites and 998 (21.7%) unique G-to-A variation sites . The signal to noise ratio for A-to-G variations is 1.67. If combined with G-to-A variations, then the signal to noise ratio would be as high as 5.32. Among the 2878 unique A-to-G sites, 797 and 2391 sites were found in the two SRR samples, respectively, and 310 sites were overlapped.The nearly nine thousand (non-unique) variation events shown in the above section belong to 4604 unique variation sites. Most of the 4604 unique sites have less than 10 reads supporting the alternative allele . This result confirmed that the prevalent A-to-G variants would not be affected by any filtering cutoffs.Since these results came from the mapping and variant calling procedures without any filtering criteria, we think it is necessary to see whether the patterns are sensitive to any filtering parameters. We re-did the analysis by requiring mapping quality > 25 and base quality (controlling for sequencing errors) > 35. We found that the number of unique variation sites (3129) slightly declined but the majority  of which are still A-to-G variations . Note that this depletion of G is statistically significant under Chi-square tests with the null hypothesis of equal numbers of A, C, G, and T . In contrast, the G-to-A variation sites did not have such a key validation feature . This consolidated our assumption that these A-to-G variations are RNA modification sites.We also wish to prove the reliability of the putative A-to-G modification sites from another angle. The base context of the A-to-G variation sites showed an obvious depletion of G upstream to the putative A-to-G modification sites  nor the deamination rate.In addition, the deamination process also obscures the inference of ancestral state. If it is confirmed that the SARS-CoV-2 has been transferred from patient No.1 to patient No.2, then one might consider the RNA-seq from patient No.1 should be the ancestral state. However, the viral RNAs in patient No.1 would undergo A-to-G modification by the hosts. The deamination enzyme only modifies a fraction of the total viral RNAs so that in patient No.1 there is still a mixture of A-version and G-version RNA reads. Technically, one could not know whether this is a polymorphic site in the virus population or it is modified by the host’s enzyme. This uncertainty makes it difficult to define the ancestral state.There is a less important but unsolved question that we think the G-to-A variation sites could also be the A-to-G modification on RNA of the “reference genome”. However, from the base context of the G-to-A sites, they did not seem to be authentic A-to-G modification sites. This strange pattern remains an open question.In summary, for many studies that claimed the optimization in variant calling pipeline, they only improved the accuracy of the alignments. Even an alignment is manually verified, we still do not know whether the A-G mismatches in the alignment should be SNPs or RNA modification sites. We appeal that this issue should be seriously discussed in the studies involving RNA viruses like SARS-CoV-2.The technically indistinguishable RNA modifications and SNPs of SARS-CoV-2 have complicated the situation where the researchers intend to reveal the evolutionary patterns behind the mutation spectrum. This is not a problem for DNA organisms but should be seriously considered when we are investigating the RNA viruses.


SHORT ROOT (SHR) RNA undergoes in cells. We found that an RNA G-quadruplex (GQ) forms in SHR mRNA and is capable of triggering RNA phase separation under physiological conditions, suggesting that GQs might be responsible for the formation of the SHR phase-separation-like phenomenon in vivo. We also found the extent of GQ-triggered-phase-separation increases on exposure to conditions which promote GQ. Furthermore, GQs with more G-quartets and longer loops are more likely to form phase separation. Our studies provide the first evidence that RNA can adopt structural motifs to trigger and/or maintain the specificity of RNA-driven phase separation.Liquid–liquid phase separation plays an important role in a variety of cellular processes, including the formation of membrane-less organelles, the cytoskeleton, signalling complexes, and many other biological supramolecular assemblies. Studies on the molecular basis of phase separation in cells have focused on protein-driven phase separation. In contrast, there is limited understanding on how RNA specifically contributes to phase separation. Here, we described a phase-separation-like phenomenon that  Liquid–liquid phase separation (LLPS) phenomena, with two liquid phases coexisting in one system, have been recently observed in the cells and shown to be responsible for organization of many complex biochemical reactions temporally and spatially . Thus, LCLN3 and BIN2 RNAs determine the identity of droplets formed by RNA binding protein, whi3, by uncovering or masking its interaction regions  proteins with well-defined domains interact with stereospecific interaction surfaces to form oligomers ; (b) pro regions . Furtherproteins .RNA has generally been considered as a regulatory component in protein-driven phase separation . HoweverSHR mRNA, an RNA important in plant development, that was observed to form an RNA phase-separation-like phenomenon in vivo. We found that SHR contains a G-quadruplex (GQ), a specific RNA tertiary structure motif. G-quadruplexes are composed of G-tetrads formed by Hoogsteen hydrogen bonding and stabilized by π-π stacking interactions and the coordination of cations, especially K+  and SHORT ROOT (At4g37650) were designed using the online program Stellaris Probe Designer version 2.0 from LGC Biosearch Technologies (see Arabidopsis roots as previously described (l-lysine slide (Thermo Fisher Scientific) and covered by a glass coverslip . The meristems were then squashed under the coverslip, before being submerged in liquid nitrogen until frozen. The coverslips were removed using a razor blade and the roots were left on the slide to dry at room temperature for 30 min. Tissue permeabilization was then carried out by immersing the samples in 70% ethanol for a minimum of one hour. Probe hybridization following removal from ethanol, slides were left at room temperature for 5 min before two washes were carried out with wash buffer . 100 ml of hybridization solution  containing SHR/SCR probes  was added to each slide. Coverslips were placed over the samples to prevent evaporation and the probes were left to hybridize at 37°C overnight in the dark. Excess hybridization solution (containing unbound probes) was pipetted off the following morning. Each sample was washed twice with wash buffer, with the second wash left to incubate for 30 min at 37°C in the dark. After wash buffer removal, 100 ml of the nuclear stain DAPI  was added to each slide and left to incubate at 37°C for 30 min. Following DAPI removal, a 100 ml 2× SSC wash was carried out before 100 ml GLOX buffer  was added to each slide and left to equilibrate at room temperature for 2 min. This was pipetted off and replaced with an anti-fade solution containing 100 ml of GLOX buffer, 1 ml glucose oxidase (Sigma) and 1 ml catalase (Sigma). The samples were then covered by 22 mm × 22 mm No. 1 coverslips , sealed with CoverGrip sealant  and imaged using a Delta Vision Elite Deconvolution microscope with a ×100 oil immersed objective (NA 1.46).Probes for gies see . smFISH OW At3g540 and SHOin vitro transcribed using Hiscribe T7 High Yield RNA Synthesis Kit . Each RNA was purified using pre-casted 6% denaturing polyacrylamide gel (Thermo Fisher Scientific) and the gel band with desired RNA product was cut and soaked in 1× TEL800 buffer  overnight, RNA was then purified with ZYMO RNA purification kit (ZYMO S1016).DNA Templates see  were ampSHR-GQ or SHR-GQm RNA was mixed with folding buffer  and denatured at 95°C for 1.5 min. For the reaction with PDS, PDS was added to a final concentration of 5 μM. For reverse transcription, 5× RT buffer , 1 μl of 5 μM Cy5-labelled primer and 0.5 μl SuperScript III reverse transcriptase (200 U/μl) (Thermo Fisher Scientific) were added. After reverse transcription, 0.5μl NaOH (1M) was added to the reaction and incubate at 95°C to eliminate RNA template. Finally, 10 μl of 2 × stopping dye solution containing 20 mM Tris–HCl, pH 7.5, 20 mM EDTA, 94% deionized formamide and Orange G was added to the reaction mixture (Orange G dye as tracker). The cDNAs were size fractionated by 8 M urea 8% denaturing polyacrylamide gel. The gel was scanned with typhoon phosphorimager and analyzed by ImageQuant 5.2.Reverse transcription stalling assay was performed as previously described . 500 ng SHR-GQ, SHR-GQm, SHR-GQsc . Samples were visualized by a confocal microscope (ZEISS LSM 880) with ×40 oil immersion objective.RNAs and Km Figure . Before 2/μ) of fluorescence intensity per pixel (pixel size 43 nm × 43 nm). In brief, for each ROI, the variance of fluorescence intensity was determined, and normalized by the mean RNA fluorescence intensity in the solution phase. 10 independent imaging areas (∼2500 μm2 each) were analyzed for each condition.The extent of phase separation was quantified by the index of dispersion (σSHORT ROOT (SHR) and SCARECROW (SCR), two key genes for Arabidopsis root development (in situ hybridization (smFISH). This is a technique which utilizes fluorescence-labelled antisense-probes to visualize individual RNA molecules in vivo , where the expression of SHR was induced by dexamethasone(2). Following the dexamethasone induction, we observed that the big globular-shape signals of SHR RNA appeared in cells after 2 h (SCR), indicating a phase separation-like phenomenon and this phase separation-like phenomenon is specifically associated with SHR expression.To further investigate this phase-separation-like phenomena in cells, we performed another smFISH experiment on a transgenic line with inducible fter 2 h . These bSHR mRNA displays a phase-separation-like phenomenon in plant cells. As a critical gene in ground tissue patterning and identity maintenance, SHR plays important roles in root development , where x ≥2, and a, b, c are ≥1 nt and ≤7  GQ, with the sequence motif as G2L3G2L3G2L2G2  motif, was predicted at position 1611–1626  stalling assay (+) but not lithium (Li+), and GQs may be preferentially stabilized by using both potassium and the G-quadruplex stabilizing ligand pyridostatin (K++PDS)  . If GQ i(K++PDS) . We obseSHR-GQm RNA, in which the Gs that contribute to G-quartets were mutated to As   × 10−4 M  . A titrae Figure  38,39).,39.SHR wM Figure .SHR-GQm RNA did not show any GQ spectrum signature at any concentration of K+, only giving a spectrum consistent with a random coil, suggesting a complete abolition of GQ formation   Figure . Since t ∼100 mM , it is mSHR mRNA. We performed a phase separation assay on SHR-GQ and SHR-GQm RNAs at the physiological K+ concentration (100 mM K+)  was not able to trigger phase separation   and founs Figure , D wheren Figure , E.SHR-GQ is capable of triggering the formation of phase separation , in which the GQ sequence was scrambled to avoid intramolecular GQ formation  and RNA concentration (2 μg/μl)  Figure , C. Thess Figure , indicatSHRscramble1-GQ and SHRscramble2-GQ), in which the flanking sequences of GQ were scrambled  in Region Of Interest (ROI) was used to quantify the extent of phase separation  are shown to be more dominant among all the GQ types (Arabidopsis are likely to form similar RNA-driven phase separation as SHR-GQ under physiological conditions.Our results indicate that the G3 GQs provide a better trigger for phase separation as compared to G2 GQs and provides further evidence that the GQ structure is responsible for the phase separation formation. Interestingly, we also found that the GQs with longer loop lengths tend to form more droplets as compared to those with shorter loop lengths Figure . The indparation . Consistparation , D. The GQ types . Given tGQ types , this su+ can vary between different developmental stages and stress conditions (+ concentrations from 100 mM under normal cellular conditions up to 700 mM in order to avoid cellular dehydration (+ concentration in the cell is likely to promote more GQ to form strongly, subsequently inducing more GQ-triggered RNA-driven phase separation. This GQ-triggered RNA-driven phase separation might be a regulatory mechanism for the cells to protect and/or store RNAs under the stress conditions such as drought.RNA GQs have been suggested to influence translation in the plant development ,43. In pnditions ,45. For ydration . This drSHR in vivo, providing the possibility of a new regulatory role in plant root development. It also reveals a new functional role for GQ structures, serving as a trigger for RNA-driven phase separation.In conclusion, our results provide the first demonstration of RNA adopting a specific RNA structure, e.g. GQ, to allow the formation of RNA-driven phase separation. This RNA-structure-triggered phase separation might be responsible for our observation of the phase-separation-like phenomenon of gkz978_Supplemental_FilesClick here for additional data file.


The complete genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Siena-1/2020 was obtained by Nanopore sequencing, combining the direct RNA sequencing and amplicon sequencing approaches. The isolate belongs to the B1.1 lineage, which is prevalent in Europe, and contains a mutation in the spike protein coding sequence leading to the D614G amino acid change. The complete genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Siena-1/2020 was obtained by Nanopore sequencing, combining the direct RNA sequencing and amplicon sequencing approaches. The isolate belongs to the B1.1 lineage, which is prevalent in Europe, and contains a mutation in the spike protein coding sequence leading to the D614G amino acid change. Betacoronavirus in the family Coronaviridae. The virus was isolated at the University Hospital of Siena  in April 2020, from a nasopharyngeal swab collected on 1 March 2020, and seeded on Vero E6 cells. This research was carried out according to the principles of the Helsinki Declaration, with reference to the document BIOBANK-MIU-2010, approved by the Siena University Hospital Ethics Committee with amendment no. 1, on 17 February 2020, regarding general data protection and regulation (GDPR).Here, we report the complete genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Siena-1/2020, belonging to the genus After 3 days, cytopathic effect appeared on the cells, and the culture medium was collected and frozen at −80°C. Since this was the first SARS-CoV-2 viral isolate in our region, we decided to sequence it. Total RNA was isolated using the NucliSens easyMAG system . Viral RNA was sequenced using both the direct RNA and amplicon sequencing approaches on a MinION instrument .Direct RNA sequencing was performed using the SQK-RNA002 kit (ONT). Briefly, about 300 ng of total RNA was ligated to the reverse transcriptase (RT) adapter, and the first strand was retrotranscribed using SuperScript III reverse transcriptase (Thermo Fisher); sequencing adapters were then ligated to the cDNA-RNA hybrid, and the library was loaded onto a flow cell (R9.4.1).https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn); primers were designed using Primal Scheme (https://github.com/artic-network/artic-ncov2019). Amplicon reads were mapped to the reference genome Wuhan Hu-1 (GenBank accession no. MN908947) with minimap2 v2.17 (https://github.com/nanoporetech/medaka) was used to build the consensus and call the single nucleotide variants. The reference genome Wuhan Hu-1 was edited using a Perl script, selecting variants with a quality score cutoff of 35 (https://github.com/CDCgov/SARS-CoV-2_Sequencing/tree/master/protocols/CDC-Comprehensive); nucleotide variations were also visually inspected using Tablet  and SRX8982905 (amplicon sequencing).The genome sequence of SARS-CoV-2 Siena-1/2020 has been deposited in GenBank under the accession no.


However, non-coding RNAs (ncRNAs) scattered around 3′ termini of the protein-coding genes, here referred to as terminus-associated non-coding RNAs (TANRs), have not attracted wide attention in RNA research. Indeed, whether TANRs are transcriptional noise, degraded mRNA products, alternative 3′ UTRs, or functional molecules has remained unclear for a long time. As a new category of ncRNAs, TANRs are widespread, abundant, and conserved in diverse eukaryotes. The biogenesis of TANRs mainly follows the same promoter model, the RNA-dependent RNA polymerase activity-dependent model, or the independent promoter model. Functional studies of TANRs suggested that they are significantly involved in the versatile regulation of gene expression. For instance, at the transcriptional level, they can lead to transcriptional interference, induce the formation of gene loops, and participate in transcriptional termination. Furthermore, at the posttranscriptional level, they can act as microRNA sponges, and guide cleavage or modification of target RNAs. Here, we review current knowledge of the potential role of TANRs in the modulation of gene expression. In this review, we comprehensively summarize the current state of knowledge about TANRs, and discuss TANR nomenclature, relation to ncRNAs, cross-talk biogenesis pathways and potential functions. We further outline directions of future studies of TANRs, to promote investigations of this emerging and enigmatic category of RNA. The encyclopedia of DNA elements (ENCODE) project aims to reveal functional elements of the human genome, thereby providing new insights into gene and genome functions . For insInvestigation of the full landscape of 3′ untranslated regions (3′ UTRs) across species and cell types has contributed substantially to our understanding of their biogenesis and functions. Studies on the functions of 3′ UTRs focused primarily on their role in the regulation of gene expression, including mRNA trafficking, translational control, metabolism, and mRNA-protein structures . HoweverFrom a technical perspective, transcriptome sequencing and microarrays show limitations for the discovery of TANRs. Indeed, transcriptome sequencing requires the construction of cDNA libraries and TANRs are often discarded during the rRNA removal step of this process or mixed with annotated transcript fragments afterward. In fact, as mixed fragments, they can partially or completely overlap with the annotated transcripts. Overlapping RNAs can be mapped as part of the annotated transcripts, alternative UTR regions, or even discarded. Furthermore, if some TANRs do not overlap with annotated transcripts, these would be filtered out as erroneous transcripts during bioinformatic analyses. In similarity, transcriptome microarrays are based on available information on annotated transcripts, usually excluding TANRs. Hence, TANRs have been mostly ignored in gene expression studies, given their lack of specific patterns, the uncertainty of their transcriptional origin, and other methodological difficulties .Although TANRs usually are not identified as high-value targets, increasing evidence implied that they are important molecules for several cellular activities. For instance, the detection of diverse TANRs in eukaryotes suggested that they are widespread, abundant, and conserved. Moreover, studies of their biogenesis and functions pointed at TANRs as versatile molecules regulating gene expression. Since the biogenesis and functions of many TANRs are still unclear, and an increasing number of TANRs have been reported, elucidating their biological functions and mechanisms of action has become a new frontier in the field of RNA research.Applications and breakthroughs of next-generation sequencing (NGS) and gene array in transcriptomics have revealed eukaryotic genomes can generate a multitude of diverse RNA species . Owing tArabidopsis thaliana, TASR peaks were identified on 287 protein-coding genes, demonstrating that TASRs mainly accumulated in leaves and young seedlings  proteins are highly specialized binding small RNAs and can regulate gene expression at both transcriptional and posttranscriptional level by interacting with other proteins . By sequof mRNAs . Such TTof mRNAs . Compare Lindner . In addi Lindner . Given tRegarding the genomic location of these ncRNAs, TASRs, aTASRs, TTSa-RNAs, and uaRNAs are located within 3′ UTRs. In particular, TANRs and aTASRs start from poly(A) signal sites, while TTSa-RNAs end at the cleavage sites. Furthermore, TALRs and a small subset of antisense CUTs/SUTs usually overlap with 3′ UTRs. On the other hand, TASNRs are located downstream of 3′ UTRs. As indicated by their name, aTASRs and antisense CUTs/SUTs are located on the antisense strand, while other ncRNAs are located on the sense strand . AccordiStudies of the biogenesis of ncRNAs are required to elucidate their functions and potential roles in the regulation of gene expression . MiRNAs In the same promoter model of TANR biogenesis, firstly, the transcription of TANR precursors is coupled to that of the upstream mRNAs using the same promoter. Then, maturation of TANRs occurs by posttranscriptional cleavage. Considering that the maturation processes of TASRs, TASNRs, TTSa-RNAs, TALRs, and uaRNAs share many characteristics, we summarize them altogether.Terminus-associated small RNAs are located within the 3′ UTR of genes where no histone modifications marking active promoters or enrichment for RNA polymerase II (RNAPII) occupancy are found . Hence, rpl26-snR49 and rpl29-snR93) highly overlapped with upstream mRNAs; no promoters were detected between mature TASNRs and their precursors; and promoter deletion analysis confirmed that the precursor of TASNR snR49 and the corresponding upstream mRNA used the same promoter for the regulation of their transcription. Thus, TASNRs undergo processing from precursors during maturation  RdRP can perform de novo synthesis of short interfering RNAs (siRNAs) that are complementary to template RNAs  stretches at their 5′ ends that are complementary to the 3′ poly(A) tails of mRNAs , nrpd1a (RNA polymerase IVa), and nrpd1b (RNA polymerase IVb) mutants. Thus, RdRPs and RNA polymerase IV are responsible for the biogenesis of some aTASRs, even though the detailed mechanisms remain unknown (The RNA-dependent RNA polymerase (RdRP) plays a key role in RNA silencing in fungi, plants, and worms by generating double-stranded RNAs (dsRNAs) from RNA templates . In the ate RNAs . Thus, dof mRNAs . These dof mRNAs . In A. t unknown . However unknown .Saccharomyces cerevisiae. This conclusion derived from the fact that the transcriptional initiation sites of antisense CUTs or SUTs are located in nucleosome-free regions (NFRs), corresponding to promoter regions. Thus, independent transcription is the main biogenesis mechanism of antisense CUTs or SUTs  together with several proteins. Well-known functional RNP particles include snoRNA ribonucleoproteins (snoRNPs) and miRNA-AGO ribonucleoproteins (miRNPs). These RNPs contain the respective RNAs and a small set of associated proteins . Within PR) mRNA could modulate PR transcription   . In a reications .in trans to regulate cell proliferation and differentiation in the absence of corresponding protein-coding transcripts  that positively regulates the expression of PHO2 by sequestering miR-399 from its target site  were reported before 3′ UTR-associated RNAs (uaRNAs) . uaRNAs MALAT1-associated small cytoplasmic RNA (mascRNA), generated from the nascent lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1). MascRNA is located at the 3′ end of mature MALAT1, and its maturation is dependent on RNase P  of RNAPII is important for coupling mRNA transcription and processing . Indeed,FLJ11812 functions as a ceRNA by sponging miR-4459 from its target mRNAs, thereby providing a novel direction for functional studies . Therefore, TANRs that contain MREs may act as miRNA sponges, thus protecting the corresponding mRNAs from translational repression or degradation. For instance, uaRNA  studies . Further studies . However studies .Once a TANR is discovered, it is challenging to know how to study its function. Basic information, such as the abundance of related mRNAs, the secondary structure, and the subcellular localization of TANRs, aids in understanding their possible functions. For detailed functional studies, induced upregulation and downregulation of TANRs represent an appropriate strategy for primary functional studies. To achieve upregulation, overexpression or synthesis of certain TANRs represents available methods. However, for most TANRs overlapping with certain 3′ UTRs that harbor important regulatory elements, some technical issues need to be overcome to eliminate the potential impact of induced downregulation on the upstream transcripts. Currently, siRNA screens and the application of CRISPR -Cas9 to delete certain DNA regions provide useful tools for functional annotation of TANRs in a native context .In summary, the discovery of TANRs in different eukaryotes suggested that they are abundant and conserved. Moreover, studies of the biogenesis and functions of TANRs indicated that they can play important roles in different cellular activities. However, since TANRs represent a novel group of ncRNAs, their biogenesis and functions still require further research. As more information about different TANRs is being reported, their involvement in the regulation of gene expression is due to be unfolded in full and presents one more intriguing observation of the versatility of RNA function.FX and X-ML contributed to the conception of the study. W-JN and X-ML wrote the manuscript. W-JN, FX, and X-ML discussed and improved the revised manuscript. All authors read and approved the final manuscript.The 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.


Infectious long-noncoding (lnc) RNAs related to plants can be of both viral and non-viral origin. Viroids are infectious plant lncRNAs that are not related to viruses and carry the circular, single-stranded, non-coding RNAs that replicate with host enzymatic activities via a rolling circle mechanism. Viroids interact with host processes in complex ways, emerging as one of the most productive tools for studying the functions of lncRNAs. Defective (D) RNAs, another category of lnc RNAs, are found in a variety of plant RNA viruses, most of which are noncoding. These are derived from and are replicated by the helper virus. D RNA-virus interactions evolve into mutually beneficial combinations, enhancing virus fitness via competitive advantages of moderated symptoms. Yet the satellite RNAs are single-stranded and include either large linear protein-coding ss RNAs, small linear ss RNAs, or small circular ss RNAs (virusoids). The satellite RNAs lack sequence homology to the helper virus, but unlike viroids need a helper virus to replicate and encapsidate. They can attenuate symptoms via RNA silencing and enhancement of host defense, but some can be lethal as RNA silencing suppressor antagonists. Moreover, selected viruses produce lncRNAs by incomplete degradation of genomic RNAs. They do not replicate but may impact viral infection, gene regulation, and cellular functions. Finally, the host plant lncRNAs can also contribute during plant-virus interactions, inducing plant defense and the regulation of gene expression, often in conjunction with micro and/or circRNAs. Over several decades numerous classes of noncoding RNAs (ncRNAs) have been identified in all organismal kingdoms. Among the three basic groups of ncRNA are short ncRNA, such as micro RNAs (miRNAs), small interfering RNAs (siRNAs), tRNA-derived stress-induced RNAs (tiRNAs), small nucleolar RNAs (snoRNAs) and piwi-interacting RNAs (piRNAs), middle size ncRNAs, and long non-coding RNAs (lncRNAs), >200 nt in length, such as cold assisted intronic noncoding RNA (COLDAIR), HOX antisense intergenic RNA (HOTAIR), etc. ,2. In paIn plant genomes, lncRNA transcripts situate as intergenic RNAs, antisense RNAs, intronic RNAs, and non-overlapping antisense RNAs . LncRNAsLncRNAs can regulate histone modifications at the chromatin level, via interacting with or acting as scaffolds for methylation and acetylation complexes. Such epigenetic effects are well described in the case of repressive complex PRC2  or COMPAAt the transcription level, lncRNAs can act as co-factors that modulate the activity of transcription factors, e.g., during plant immune responses . LncIn this review, we focus on lncRNAs related to plant RNA viruses, both to those that do not originate from virus genomes (viroids and satellite RNAs) and those of viral origin  . VariousViroids are a group of infectious plant lncRNAs that are composed of RNA genomes and replicate by using the host enzymatic activities. Viroids carry the circular, single-stranded, non-coding RNA molecules that represent the smallest known replicons among all living objects, ranging between 246 to 401 nucleotides (nt) in length ,14. ViroPospiviroidae and the Avsunviroidae, each further divided into genera according to their RNA structures and relationships. A brief overview is given in Their mode of action along with the host processes makes the viroids unusually complex lncRNA molecules . UniquelPospiviridae replicate by asymmetric and the Avsunviridae by symmetric rolling-circle mechanisms  [Pospiviroidae the monomeric circular viroid RNAs replicate by the RCR mechanism to produce an oligomeric longer-than-unit strand, that is cleaved into replicative monomeric RNAs by one or more host ribonucleases (RNases) most likely the members of RNase III family. The recognition of the cleavage site by host RNases is determined by the stem-loop structures in the viroid RNAs around the CCR region. Thus produced monomeric linear (ml) RNA serves as a template for the synthesis of the ml (+) strand RNA [Both families of viroids adopt the rolling-circle replication (RCR) mechanism, but the chanisms . Pospiviroid RNA . Studies5 (RPL5) ,19,20. Irand RNA . The ml rand RNA ,23.Avsunviroidae replicate in the chloroplasts by the symmetric RCR mechanism, where (+) stranded circular viroid RNAs are copied into the oligomeric (-) RNAs that are cleaved and ligated to the mc (-) strands. The mc (-) strands are used as a template to undergo a second round of the RCR to produce mc (+) RNA [Avsunviroidae [2+ beyond physiological conditions questioned the feasibility of the reaction in vivo [The members of  (+) RNA . The nuc (+) RNA ,25,26,27viroidae ,29. Besi in vivo . However in vivo .The extensive secondary structure plays a critical role in the viroid life cycle, such as in host plant invasion, replication, pathogenesis, and transport. Viroid RNA structures have been studied in great detail by using SHAPE analysis whereas their structural 3D complexity has been confirmed via the direct visualization of single-RNA molecules by atomic force microscopy ,32. StruN. benthamiana plants, signifying that this viroid should be viewed as a functional lncRNA [Viroid infections often cause disease symptoms in such important crops as apple, avocado, coconut, grapevine, hop, peach, potato, tomato, and others . Recent l lncRNA .Viroids have emerged as productive tools with which to study the interactions not only of their replicable lncRNAs, but also the plethora of functions of host lncRNAs in general . Currentde novo origin of viroid-like replicons via a parsimonious scenario. From the pool of various RNAs in eukaryotic cells, some can circularize and serve as seeds of the process [Among several hypotheses regarding the origination of viroids, Kiefer et al. (1983) suggested that they might come from transposons or retroviruses . However process .Coleus blumei infected with coleus blumei viroids (CbVds). Based on the NGS data it has been revealed that the central conserved region (CCR) of the viroid is pivotal in the biogenesis of sRNAs for both host RNA silencing and the genome replication of the viroids [NGS has been widely used in viroid research for the discovery of new viroids and diagnostic purposes. The technology has been employed to analyze viroid sequences and host gene expression (transcriptomic) in response to viroid infection . Progres viroids . Deep se viroids . Similar viroids . TherefoDefective (D) and defective interfering (DI) RNAs represent of the class of lncRNAs that are derived from the helper RNA virus genome by one or more premature termination and reinitiation events, resulting in functional replicative templates . The repCurrent analytical developments, especially in NGS sequencing, have progressed rapid advancements in the understanding of the mechanisms of the generation of DI RNAs, and their role as signals promoting antiviral immunity during infection, affecting viral dynamics and evolution during host-virus interactions. The diversity of defective RNAs is much larger than initially detected and they appear to be present in virtually every virus population. The use of single-cell sequencing technology and bioinformatics detected jumbled or rearranged viral sequences, and their populations are not the same if generated in different hosts . TruncatCucumber necrosis virus, Brome mosaic virus, Turnip crinkle virus, Cucumber mosaic virus, Bovine viral diarrhea virus, and Hepatitis C virus have shown that RNA breaks, strong hairpin structures, or that AU–rich stretches enhance the replicase in order to switch the template between the donor RNA and the acceptor RNA, leading to mosaic or truncated progeny RNAs, with loss in peptide coding functionality, but retaining the ability to replicate [de novo randomly due to faulty viral proteins such as mutated RdRp, nucleoprotein variants, and other structural proteins that affect replication and recombination, as illustrated in Defective RNAs generally consist of the non-contiguous portions of the helper virus genome, synthesized by the viral RNA-dependent RNA polymerase (RdRp). The replication-dependent template-switching mechanism, either the replicase dependent or the forced template-switching mechanism, are among the well documented and popular means for synthesis of the defective RNAs . The anaeplicate ,68,69,70Crinivirus, DI RNAs comprise a mosaic of the multipartite parental viral genome [Tombusviruses [To date, DI RNAs resulting from the above mechanisms have been characterized for a variety of plant RNA viruses carrying positive-sense, negative sense, double-stranded and ambisense genomes ,60. For l genome , while dsviruses ,72. TobrPotato mop-top virus the 8K protein and the triple gene block 1 (TGB1) proteins are known to alter the biogenesis of the DI RNAs [The roles of viral replicase proteins, the replication/recombination sequence domains, host factors, and growth conditions have been found to affect the above mechanisms of defective RNA generation . The rol DI RNAs . The con DI RNAs ,83,84.XRN1 is found to promote the formation of DI-RNAs [Host factors can influence the mechanism of the DI-RNA generation. Using the yeast single gene knock out library, several dozens of host genes that could affect virus RNA recombination have been identified in tomato bushy stunt virus (TBSV). The host exoribonuclease-like  DI-RNAs ,85. The  DI-RNAs ,87. Thus DI-RNAs .Tombusviruses and Cucumoviruses has been extensively studied [de novo generation of DI RNAs has been observed for the first time in Tombusviruses, after serial passages of DI RNA-free viral RNA through host plants. The presence of a DI RNA considerably attenuates Tombusvirus infection, from lethal necrosis to persistent symptoms. In general, DI-RNAs have complex relationships with the helper virus (HV) regarding the competition for viral- and host factors, and mitigation of antiviral responses and/or disease symptoms in the host plant. D RNAs have also been reported in the tomato black ring virus (TBRV), one of the Nepoviruses. The D RNA formed by a single deletion in the RNA1 molecule interfered with the replication of TBRV. Recently, DI-RNA has also been shown to increase the vertical seed transmission of the virus [The pathogenesis of the DI RNAs in  studied ,65 and dhe virus ,90.Potato mop-top pomovirus, where regulation of virus accumulation depended on the antagonistic roles of the relative levels of 8K protein and D RNAs (the 8K gene is needed for efficient virus accumulation but D RNA impairs efficient virus accumulation) [Defective RNA-virus interactions evolve into mutually beneficial combinations. Such domestication processes enhance virus fitness, giving the helper virus competitive advantages in its relationship with the host, mostly by moderating disease symptoms . The ratulation) .Tombusviruses [The mechanism of interference is not clearly understood but the possibilities include (i) competition for transacting factors, (ii) interaction with viral products, (iii) inhibition of pathogenicity determinants, and/or (iv) activation of RNA interference . The firsviruses ,91. Funcsviruses . As for sviruses . This suRecent NGS data for animal RNA viruses demonstrate that hundreds of defective RNAs can arise within a single viral infection, but only some are repeatedly detected in different samples . More reThe lack of sequence homology to the helper virus distinguishes defective RNAs from sequence-unrelated satellite (sat) RNAs, although they are also dependent on the helper virus . In contBamboo mosaic virus satRNA, which encodes an RNA-binding protein [Nicotiana tabacum, a large amount of siRNAs derived from Y-sat RNA shows yellowing of leaves caused by the down-regulation of chlorophyll biosynthetic gene ChlI by RNA silencing [Nicotiana benthamiana plants co-infected with the satRNAs showed a higher fold change of differentially expressed genes (DEGs) involved in the biosynthesis of secondary metabolites, translation, ribosome biogenesis, and RNA metabolic processes [As far as their general organization, the satRNAs include three categories of single-stranded (ss) RNAs: large linear ss RNAs (0.7–1.5 kb) that encode a protein, small linear ss RNAs (less than 0.7 kb), and small circular ss RNAs. An example of the first category is  protein ; however protein . This we protein ,101. The protein . The sys protein , whereas protein . In Nicoilencing ,105. Therocesses .Zhu et al. (2011) suggest that satRNA attacks CMV RNA via RNA silencing , leadingInterestingly, the third category of small circular satRNAs is called virusoids, because they are similar to viroids but are dependent upon the helper virus . The cirThe structures of satRNAs are known to be conserved and are required for successful replication. For example, in lucerne transient streak virus satellite RNA (LTSV satRNA), most of the mutations reverted to the wild type by 21 days post-inoculation (dpi) and had wild-type secondary structures . Thus, sPlant RNA viruses generate subviral RNAs  for high-yield translation of downstream ORFs. sgRNAs can be produced by internal initiation from the subgenomic promoter, by premature termination, leader primed transcription, or discontinuous transcription. BMV, one of the most commonly studied plant RNA viruses, transcribes sgRNA4 from an internal subgenomic promoter in the RNA3, which is translated into the coat protein . By the Benyviridae, the beet necrotic yellow vein virus generates a non-encapsidated 500 nt lncRNA (RNA3sub) of yet to be established function [However, selected viruses produce non-replicating RNAs that do not encode any protein, often by incomplete degradation of genomic RNAs. Such degradation can be due, e.g., to cellular 5′ to 3′ XRN1 exonuclease, until it stops at a structured element. Such lncRNA products may then interact with different cellular factors used for transcription, translation, splicing, or RNA interference ,124,125.function .Among common features shared by virally-derived lncRNAs are their highly stable structures, the use of RNAPII or RNA degradation mechanisms for their production, their importance in the infection cycle, and their direct intervention in gene regulation. This can modulate the hormonal and metabolic signaling pathways to enhance viral infection. For example, the citrus tristeza virus (CTV) produces a lncRNA called low molecular weight tristeza 1 (LMT1), which is associated with maintaining virus accumulation, movement, and infectivity by lowering the production of salicylic acid (SA) and reactive oxygen species (ROS) required for antiviral defense . Thus, vRegarding the origination of plant lncRNAs, one theory links their biogenesis to TE elements ,132. BecN. benthamiana [Studies have been conducted to evidence the contribution of host lncRNAs during plant-pathogen interactions . For insthamiana . Host lnIn another work on the TYLCV-tomato system aiming at the identification of lncRNAs as key regulators of gene expression, the NGS RNA-seq revealed different patterns of lncRNAs and circRNAs in infected vs. uninfected susceptible plants . The silSeveral decades of extensive research on viroids have contributed significant progress toward the understanding of interactions of vd-RNAs with plant cell components, especially to explain the involvement of vd-sRNA in viroid-induced disease symptoms and induced gene expression, and the role of RNA silencing. However, further analyses are required on the mechanisms of viroid-induced host gene expression, as well as on the contribution of circRNAs in the regulation of translational machinery. Structure prediction techniques will further the study of viroid biology, especially concerning means of escape from RNA silencing surveillance.Likewise for D RNAs, recent NGS sequencing projects have revealed considerable diversity among D RNAs in plant viruses. Nevertheless, some questions remain, including the detailed molecular mechanisms of the D RNA biogenesis, their control of host-virus pathogenesis, the degree to which D RNA populations contribute to virus evolution and host range, and how the interactions of D RNAs with viral and/or host factors affect the stages of the virus life cycle or induction of symptoms.As for host plant lnc RNAs and plant responses to pathogens/viruses, although a great number of host- and pathogen-related lncRNA has been discovered in many organisms, and considerable efforts have been undertaken to recognize lncRNA-mediated plant defense against viruses, the precise antipathogenic defense remains largely obscure, e.g., in terms of the link to the RNA silencing machinery. In general, lncRNAs have been found to play a role in the modification of chromatin architecture, and to interact with transcription and translation factors, with proteins of signaling cascades, or with other cell host factors. These lncRNAs could likely act to overthrow cellular defense systems. Understanding the relevance of lncRNAs to disease in terms of virulence development and anti-viral immunity will change our views on RNA regulations and will facilitate the designing of new antiviral strategies.