Introduction: Eslicarbazepine acetate (ESL) is an antiepileptic drug approved as monotherapy or add-on for the treatment of epilepsy with seizures of focal onset. ESL owns a good profile in terms of efficacy and tolerability, but its effects on EEG activity and connectivity are unknown. The purpose of this study was to investigate EEG activity and connectivity changes after ESL treatment in persons with focal epilepsy (PFE).Material and Methods: We performed a multicentre, longitudinal, retrospective, quantitative EEG study on a population of 22 PFE, and a group of 40 controls. We investigated the ESL-related changes of EEG power spectral activity and global connectivity  for standard frequency bands (delta to gamma). Seizure frequency was evaluated to assess ESL efficacy in our cohort.Results: ESL significantly enhanced both global power spectral density and connectivity for all frequency bands, similarly for all connectivity measures. When compared to the control group, Post-ESL power was significantly higher in theta and gamma band. Pre-ESL connectivity values were significantly lower than control for all frequency bands. Post-ESL connectivity increased and the gap between the two groups was no longer significant. ESL induced a 52.7 ± 41.1% reduction of seizure frequency, with 55% of clinical responders (reduction of seizures ≥50%).Discussion: ESL therapy induces significant enhancement of brain activity and connectivity. Post-ESL connectivity profile of epilepsy patients was similar to the one of healthy controls. Eslicarbazepine (ESL) is an antiepileptic drug (AED) approved for the treatment of epilepsy with seizures of focal origin . ESL is The molecular mechanism on which ESL relies to express its therapeutic action is mainly related to the inactivation of voltage-gated sodium channels, similarly to oxcarbazepine, and carbamazepine . This meVery little is known on the effects of this medication on the brain cortex as a system. AEDs have an effect on brain networks and although the literature is poor of studies explicitly addressing this topic, there is plenty of evidence that antiepileptic medications influence cortical rhythms and networks as measured by EEG –14.EEG is a sensitive and reliable tool to substantiate cortical function. In particular, each frequency band power  owns a functional significance. In epilepsy patients, the study of rapid rhythms can help in better understanding positive AED effects of cognition, attention, perception and language networks. On the opposite, the study of slow frequency bands allows to detect effects on memory, drowsiness and cortical plasticity [for a review see Assenza et al. ]. In a rHere we have performed a multicentric, longitudinal study to investigate the impact of ESL on cortical activity and connectivity, as explored by quantitative EEG, in a population of persons with seizures with focal onset (PFE).p > 0.200 consistently) were enrolled at the epilepsy clinic of Department of Human Neurosciences enrolled of Policlinco Umberto I Universitary Hospital of Rome and of Campus Bio-Medico University of Rome. The study was approved by the ethic committee of Policlinco Umberto I Ethic Board—Rome—and Campus Biomedico University Ethic Board—Rome-. All patients signed a written informed consent. All procedures were performed in agreement with the 1964 Helsinki declaration and its later amendments.Twenty-two PFE ; 46% females) and a control group of forty healthy subjects  ESL assumption and after at least 3 months of ESL assumption;- resting state EEG free of relevant artifact > 5 min.Inclusion:- PFE taking neuroactive drugs other than antiepileptic drugs;- antiepileptic medication titration in between the two EEG recordings.- Clinical seizures in the 24 h before EEGsExclusion:Healthy subjects were enrolled among the relatives of patients investigated at the outpatient clinic, hospital personnel, and volunteers and were interviewed by a neurologist to rule out medical conditions potentially biasing the study. Patients underwent standard clinical EEG recording before starting eslicarbazepine acetate and after a 3 months period of follow-up. Detailed clinical info is reported in Table Nineteen channel-EEG was acquired with a Micromed recorder  between 11:00 and 13:00 a.m. and between 16:00 and 18:00 p.m to minimize drowsiness. The electrodes were placed according to the international 10–20 system . The reference was placed on FPz and the ground on FCz. Impedance was kept below 5 kOhm for all electrodes. The sampling rate was set to 256 Hz. Eye blinks, eye movements and electrocardiogram (EKG) were recorded using dedicated bipolar electrodes. The resting EEG recording lasted 5 min and was performed with patients with closed eyes, seated on a comfortable armchair in a quiet room. Patients were instructed as follows: “Free your mind, do not think about anything and just relax.” Subjects were also asked to keep their regular wake/sleep cycle before participation. Pre ad Post medication EEGs were measured always using the same apparatus.https://sccn.ucsd.edu/eeglab/ and http://neuroimage.usc.edu/brainstorm). EEG pre-processing consisted of: (a) visual inspection for rejection of possible electrographic seizures, interictal epileptic activity, bad channels, (b) [1–70Hz] band-pass filter, (c) [50Hz] notch filter, (d) Independent Component Analysis (ICA)   to removis (ICA) , 21.Epochs containing interictal epileptic activity  were rejected. Those PFE with one EEG recording containing more than 20% of interictal epileptic activity were excluded .To assess the effect of ESL on brain networks we performed measures of resting state brain activity and connectivity. As measure of activity, we computed the Power Spectrum Density (PSD) by standard FFT approach (Welch procedure: average of non-overlapped windows with a duration of 2 s) for the following frequency bands . To obtain a measure of global activity, we averaged PSD measures over all channels.As for connectivity, we focused on the Phase Locking Value (PLV). Phase Locking Value is a very popular measure of non-directional frequency-specific synchronization reflecting long-range integrations. It assesses the extent to which the phase difference between two signals changes over time and is a measure of cortical synchronization based on phase covariance between signals. PLV owns a good time resolution and provides insight on the synchronization of fast changing electrophysiological dynamic. High PLV indicates higher synchronization –27. We mThe low number of sensors and the detrimental effect of volume conduction on estimation of EEG connectivity might be an issue when assessing connectivity between sensors or brain regions . TherefoESL efficacy was measured on the percentage of seizures reduction. We defined as responders those patients with seizure reduction ≥50% and non-responders the remaining ones.As EEG activity is very sensitive to sleepiness and cognitive performance , we searBand  and Time (two levels: Pre-ESL and Post-ESL) as within subject factors. In order to compare the epilepsy group and the control group, we applied a mixed model repeated measure ANOVA, with Band  as within subject factor and Group two levels: Control and Pre-ESL (or Post-ESL) as between subjects factor. The Greenhouse-Geisser correction was applied when needed. A paired t-test was used to study changes of behavioral scale scores. The significance level was set to alpha = 0.05.Statistical analysis was performed using the IBM SPSS Statistics (Ver. 24) and Matlab (Mathworks). Data distribution was checked by means of Kolmogorov-Smirnov test. The effect of ESL on global cortical power and connectivity within the epilepsy group was tested with a repeated measure ANOVA with Time , suggesting a global increase of connectivity after ESL. No significant Band by Time interaction was found , ruling out a specific ESL effect for selected frequency bands  = 1077.993, p < 0.001] was also found, suggesting an expected difference in power across bands.The repeated measure ANOVA showed a significant main effect of the factor Time  related to a global increase of connectivity after ESL  = 0.196, p = 0.881], ruling out a specific ESL effect for selected frequency bands. A significant Band main effect  confirmed the expected connectivity modulation over frequency bands. Similar results were found on different measures of brain connectivity. For AEC a significant factor Time , together with lack of Band by Time interaction, suggested a global higher connectivity level Post-ESL as compared to Pre-ESL. A similar trend, although without reaching statistical significance, was revealed by the analysis of the orthogonalized version of AEC   = 0.695, p > 0.200], but a significant Band by Group interaction . The post-hoc tests, however, did not reveal any significant difference after Bonferroni correction. The comparison between Post-ESL and controls also revealed a significant Band by Group interaction  related to a significantly higher Post-ESL power for Theta and Gamma bands (Bonferroni-corrected post-hoc comparisons p = 0.018 and p = 0.06 respectively).The comparison between Pre-ESL and Controls revealed a significant factor Group  and no interaction, suggesting that PLV connectivity was significantly higher in the controls as compared to epilepsy patients before ESL administration. Notably, no significant Group difference nor interactions were found when comparing Controls vs. Post-ESL , suggesting that ESL reduced the gap between the epilepsy group and controls for all frequency bands  = 22.815, p < 0.001, Group by Band interaction p > 0.200; Orthogonalized AEC: Factor Group F = 10.281, p = 0.002, Group by Band interaction p > 0.200], whereas the comparison between Controls and Post-ESL did not reveal any significant Factor Group or Group by Band interaction   and an unchanged SSS (p = 0.08). Depression values were also ameliorated after ESL  with respect to the Pre.Twelve patients responded to ESL  after an average period of treatment of 83.1 ± 19.6 days. None of our patients increased the seizure frequency. The mean seizure reduction was 53.2 ± 42.6% . In the subgroup of patients with available behavioral data, after ESL, sleepiness assessment showed an improvement of ESS  and behaves potentially better than carbamazepine and topiramate, which selectively increase delta and theta bands, reduce faster cortical rhythms and more often result in cognitive impairment , 13.Very recently the study of brain connectivity and networks has led to a new conceptualization of focal epilepsy, showing that it is neither involving the entire brain nor a single cortical spot, rather specific brain networks . EpileptFuture studies should rely on a sample size sufficiently large to allow the characterization of the topographical distribution of the effects in relation to the location of the epileptic focus and to other determinant variables, such as: etiology, disease duration, age . This exIn conclusion, in this proof-of-principle study we demonstrated that ESL treatment produced an increase in global cortical power and connectivity and encourage the study of quantitative EEG as a potential tool for assessing epilepsy and the effects of antiepileptic medications.GP, OM, PP, MT, VD, and GA: Study design; OM, PP, MT, LR, JL, MB, CD, and GA: Patients recruitment; GP, MT, VD, and GA: Data analysis; GP, GA, OM, VD, and MT: Data interpretation; GP, OM, and GA: Manuscript preparation; All: Manuscript revision.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.


The pre-mRNA of the Epstein–Barr virus LMP2 (latent membrane protein 2) has a region of unusual RNA structure that partially spans two consecutive exons and the entire intervening intron; suggesting RNA folding might affect splicing—particularly via interactions with human regulatory proteins. To better understand the roles of protein associations with this structured intronic region, we undertook a combined bioinformatics (motif searching) and experimental analysis (biotin pulldowns and RNA immunoprecipitations) of protein binding.Characterization of the ribonucleoprotein composition of this region revealed several human proteins as interactors: regulatory proteins hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP U, HuR (human antigen R), and PSF (polypyrimidine tract-binding protein-associated splicing factor), as well as, unexpectedly, the cytoskeletal protein actin. Treatment of EBV-positive cells with drugs that alter actin polymerization specifically showed marked effects on splicing in this region. This suggests a potentially novel role for nuclear actin in regulation of viral RNA splicing.The online version of this article (10.1186/s13104-019-4070-1) contains supplementary material, which is available to authorized users. EBV is olecular –10 and iructures , 12, whiructures . In addiructures ; a proceructures . The splThe unusual stability and conservation of this fold  suggestsTo identify putative protein interactors we utilized the program RBPmap to scan the 119 nt LMP2 intronic RNA structured region for human regulatory protein binding motifs ; completWe also tested several other RNA-binding proteins that are predicted to be direct interactors or that might have protein–protein associations with HuR, hnRNP U or hnRNP A1. Although hnRNP L had a predicted binding site on the intronic structure, RIP data did not show any evidence of interaction , and 10% FBS  was used. Cells were maintained in a humidified incubator at 37 °C and 5% CO2.Human Burkitt lymphoma B cell lines were generous gifts from Joan Steitz ) and Siegfried Janz . For cell culture work, RPMI growth medium supplemented with 2 mM The template for in vitro transcription of the full-length RNA was generated by PCR amplification. The oligonucleotides for HP4 template was purchased from IDT and the double stranded template was generated by heating to 95 °C then slow cooling to RT. The biotinylated RNA was synthesized using the T7 MAXIscript transcription kit (Invitrogen). After incubation, samples were DNase treated then purified by Phenol/Chloroform extraction.2) was added and the RNA allowed to incubate at RT for 20 min. BJAB cells (~ 107) were harvested and resuspended in 200 µl of RIP lysis buffer . The cell suspension was sonicated briefly and 800 µl of RIP correction buffer  was added along with protease and RNase inhibitors (Thermo Scientific). The lysates were then nutated with the folded RNA for 1 h at 4 °C. Pre-cleared Pierce Streptavidin Agarose beads (50 µl) were added and nutated for another 1 h. Beads were spun down at 1000×g (2 min at 4 °C) and washed 3 times with 700 µl of cold NET buffer .Folding of the biotinylated RNA (3 µg) was done in a reaction volume of 45 µl (in water) by heating to 90 °C, then snap cooling on ice. After 5 min, the 10× RNA structure buffer . Gels were silver stained using a silver stain kit (Pierce) and bands of interest excised. Excised bands were sent to the Protein Facility of the Iowa State University Office of Biotechnology for analysis via LC–MS/MS.7) were washed with ice cold PBS, resuspended in 2.5 ml PBS and irradiated in a 10-cm dish on ice with 254-nm UV light at 800 mJ/cm2. BJAB-B1 and Raji cell lysates were prepared using the same method described in the previous section. The lysates were pre-cleared by nutating with 20 μl Protein A/G-Sepharose (SantaCruz sc2003) for 1 h at 4 °C. For each immunoprecipitation, 3 µg of the following antibodies were used: p54nrb/NONO (sc376865), HNRNP U (sc32315), HuR (sc5261), HNRNPA1 (sc32301), PSF (sc101137), from Santa Cruz; β-Actin (622102) from Biolegend or HNRNPL (A303-895A) from Bethyl Laboratories. Control IPs were carried out with Normal Mouse IgG (sc2025) or normal rabbit IgG . With the antibody, 20 µl of beads were added and incubated for 2 h at 4 °C. The beads were washed with NET buffer and I ml of Trizol (Invitrogen) was added to the beads. A 10% input was prepared by extraction RNA from 100 µl of lysate.BJAB-B1 or Raji cells  treated and concentrated using Zymo RNA Clean and Concentrator-5 (R1015) kit using its protocol. Reverse transcription (RT) reactions were performed with Superscript III using random hexamer primers (Invitrogen). For the RT reaction, the control RNA was diluted tenfold to prepare the 1% input. QPCR was performed using PowerUP Sybr mix and the QuanStudio3 instrument (Thermo Fisher). As the controls no-RT and no template reactions were included.5 cells) were isolated at three different time points and RNA was extracted using Trizol form the harvested cells. The gene expression levels were assessed by qPCR using the threshold cycle (ΔΔCT) method.For the actin assays latrunculin  was used in concentrations reported previously (1). Following drug treatments, BJAB B1 or Raji cells  and the timing of these associations. We have suggestive results for actin, which point to a stimulatory roles for this association in splicing of this structured intronic region. However, we do not know how widespread are the effects of disrupting actin on splicing across EBV (and human) RNAs. These limitations, however, suggest many additional future analyses to parse out the roles of RNA structure and associations in LMP2 splicing and to better understand nuclear actin’s roles in regulating viral and host mRNA splicing.Additional file 1: Table S1. RBPmap predictions of proteins predicted to bind motifs in the LMP2 structured intronic region.Additional file 2: Figure S1. Silver stained gels from biotin pulldowns. Bands P553-02 and P636-01B were identified as actin and hnRNPA with LC/MS–MS. M—Marker, L—lysate, FL—Full length 119 bp RNA construct of the structured intronic region, HP4—truncated RNA construct containing only a 25 bp sub region HP4.Additional file 3: Table S2. Mass spectrometry results on isolated silver stained bands  of independent experiments all normalized to control RIP with IgG. All RIPs (except for RIPs for NONO and hnRNP L) were carried out as either 2 or 3 independent experiments.Additional file 5: Table S3. STRING analysis of validated protein interactors of the LMP2 structured intronic region.Additional file 6: Table S4. List of primers.


Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis. Influenza defective interfering (DI) particles are defective virions that harbour large internal deletions in at least one segment of the viral genome . This loHigh-throughput sequencing revolutionizes the field of DI particle research because of its ability to identify and quantitate different DI-RNA species. Recently, several studies attempt to use Illumina sequencing as a new approach for identifying different influenza DI species . UnfortuIn this study, we observed the presence of DI-RNAs in nasopharyngeal aspirates (NPAs) from patients who were infected by H7N9 in high abundance and adopted SMRT sequencing to identify the entire sequence of these DI-RNA species. From the identified DI-RNA sequence, the highly diverse DI-RNA species generated in patients with H7N9 infection could be observed. Intra-species overlapping sequences at both ends of the large internal deletion around the putative breakpoint were noted. We have further extended our study of DI-RNA species in cell line and mouse model infected with H7N9 AH1 strain with SMRT sequencing. We also compared the identified DI-RNA species from infected mouse model with both Illumina and SMRT sequencing, and found that both platforms were able to identify the common large internal deletion breakpoint but SMRT could also detected DI-RNA species with multiple breakpoints owing to its long sequencing read length. While the exact mechanism of the generation of the DI-RNA by influenza has not been revealed, SMRT appears a promising tool for characterizing DI-RNA entities in future mechanistic studies.Two influenza A viruses, A/WSN/1933 [H1N1] and A/Anhui/1/2013 [H7N9], were used in this study. H1N1 (WSN) virus was generated using pHH21-plasmid reverse genetics system . H7N9  (UW 13–372). Six NPAs of 10–76-year-old Chinese patients with H7N9 infection were collected in Hong Kong during 2014–2017 and cryo-preserved. NPAs of four Chinese patients with H3N2 infection were collected in Hong Kong during 2017.TM medium (Gibco Cat# MEPI500CA) supplemented with EpiLifeTM Defined Growth Supplement (EDGS) (Gibco Cat# S0125) and Keratinocyte-SFM Medium (Gibco Cat# 17005042), including the Keratinocyte Supplements which contain bovine pituitary extract and recombinant EGF.Human normal bronchial epithelial cells (NHBE) were purchased from Lonza (Cat# CC-2540). NHBE were cultured in 1:1 ratio of EpiLife2. Cells were then washed twice with blank RPMI 1640 and cultured in RPMI 1640 medium supplemented with TPCK-trypsin (Sigma). Total cellular RNA was extracted at 24 h post-infection.NHBE cells were seeded in six-well plates and infected with H1N1 or H7N9 at multiplicity of infection (MOI) of 0.5 for 1 h at 37°C supplemented with 5% CO4.8 PFU [3 PFU of H7N9 virus. The mouse lung tissues and homogenates were collected on 2 and 4 days after virus inoculation for the quantitation of DI-RNA.All animal experiments were followed to the standard operating procedures approved by the University of Hong Kong committee on the use of live animals in teaching and research (CULATR: 3536-14). Six- to eight-week-old female BALB/c mice were obtained from the Laboratory Animal Unit of the University of Hong Kong. Virus challenge experiments were performed in biosafety level 2 (WSN virus) and 3 (H7N9 virus) animal facilities. Mouse 50% lethal dose (LD50) was determined with serial dilutions of H7N9 virus by intranasal inoculation as previously described . The LD54.8 PFU . Group oViral RNA was extracted from NPA of H7N9-infected patients and H7N9-infected NHBE using a QIAamp Viral RNA Mini kit (Qiagen) following the manufacturer’s instructions. Total RNA was extracted from virus-infected NHBE cells and mouse lung tissues using Trizol reagent (Invitrogen) following the manufacturer’s instructions. Random hexamer primers were used to synthesize cDNA from viral or total RNA using a Transcriptor First Strand cDNA Synthesis kit . Primers designed for DNA amplification of PB1 DI-RNA detection in mice and SMRT sequencing were shown in Supplementary Table S1. The viral genome and total RNA were amplified using a PrimeSTAR® GXL DNA Polymerase (TaKaRa). PCR amplification cycled under the following condition: 95°C for 3 min, followed by 30 cycles of 98°C for 10 s, 60°C for 15 s and 68°C for 3 min. Following electrophoresis in 1% agarose gel and the relative intensity of DI-RNA and full length RNA was determined by ImageJ v1.52. All fragments within 100–1000 bp were considered as DI-RNA and fragments within 1500–2500 bp were classified as full length RNA.TM Quick Gel Extraction Kit (Invitrogen). The gel-purified product was TA-cloned into pGEM-T vector using pGEM-T and pGEM-T Easy Vector Systems (Promega) which was then plasmid-extracted with PureLinkTM Quick Plasmid Miniprep Kit (Invitrogen) and subjected to Sanger sequencing. Sequences of eight individual clones were aligned to the full length PB1 sequence.To perform Sanger sequencing on the common DI-RNA isolated from the PB1 segment of H7N9-infected patient NPA sample, its PCR product was purified using a PureLinkOne of the H7N9-infected mice was selected for identifying DI-RNAs using Illumina sequencing. The Illumina sequencing and library construction was performed by Novogene. In brief, NEBNext® Ultra RNA Library Prep Kit was used for library construction. After adapter ligation, 10 cycle of PCR amplification was performed for sequencing target enrichment. Sequencing was performed with 150 bp paired end reads on an Illumina NovaSeq 6000. FASTQ sequences of each sample were aligned against the H7N9 (A/Anhui/1/2013) sequence using TopHat2  with 5–1TM Template PreP Kit 1.9-SPv3. One Sequel SMRT cell was used to sequence 7 amplicons. The raw reads were separated and circular consensus sequence (CCS) reads were generated using SMRT Link v5.0.1. The SMRTbell adapter sequences were removed and CCS reads within 150–2400 bp were selected for further analysis. The histograms of numbers of counts and length of DI-RNA sequence were generated using R [The remaining PCR products  were cleaned up using GeneJET Gel Extraction and DNA Cleanup Micro Kit (Thermo Fisher). The second round DNA amplification was performed using KAPA HiFi HotStart PCR kit (Roche) with Barcoded Universal Primers (Pacific Biosciences). The barcoded PCR products were pooled for the SMRTbell library construction using SMRTbell using R . The seqDI-RNAs are most frequently identified in polymerase genes PB2, PB1, and PA, but not in nucleoprotein NP . Therefo3 PFU of H7N9 (AH1) viruses. High amount of DI-RNA was detected in H7N9-infected lung tissues as early as days 2 and 4 post-infection  was selected for examining the generation of DI-RNAs in cell culture and mouse model. Since H1N1 WSN strain has been reported to generate DI-RNAs in infected cultured cells with available sequence information , we compnfection B. The paTo gain a more comprehensive understanding on the variety of DI-RNA species, high-throughput sequencing was applied to detect and quantity the abundant of different DI species. Currently, the sequencing platform used for identifying internal deletion of influenza genome is Illumina sequencing ,24. As tSMRT sequencing was applied for the following experiments due to the identification of rare DI-RNA species and receiving more comprehensive information. The raw reads of SMRT sequencing for all H7N9-infected individuals were listed in Supplementary Table S2. The three NPAs of H7N9-infected patients with the highest sample quality and viruses-infected NHBEs were selected and subjected to SMRT sequencing. Consistent with the DI-RNA pattern observed in gel electrophoresis, high abundance of DI-RNAs in PA, PB1 and PB2 was revealed in SMRT sequencing but relatively less abundant for NP segment . In-deptAfter aligning DI-RNA sequences to the referenced sequences, similar observation was invariably resulted from SMRT sequencing of H7N9-infected NHBEs while DI-RNA was minimally detected in H1N1-infected cells by contrast , which hThis study demonstrated the robustness of SMRT sequencing in identifying the DI-RNA species. SMRT sequencing could successfully demonstrate the diversity of DI-RNA species and the relative abundance of each species. With the long-read length of SMRT sequencing, complete information of each DI-RNA could be obtained due to no fragmentation of DI genome required. For example, long overlapping sequences at the breakpoint region, and most importantly, DI-RNAs with multiple internal deletions were identified. In addition, the influenza DI-RNAs have been reported in cultured cells with influenza B virus infection  and H1N1The introduction of Sanger sequencing revolutionizes the field of DI particles due to identification of the breakpoint location for a single DI genome. Since then, Sanger sequencing becomes a traditional and standard sequencing platform for detecting and identifying DI-RNA species. Recently, NGS has been introduced and several studies attempt to use this new approach for identifying different DI species ,11,12. Ain-vitro infection experiment in further studies.With the ability to obtain long-read length by SMRT sequencing, the comprehensive information of DI-RNA species could be obtained from both H7N9-infected clinical specimens and primary culture cells. From the high-throughput sequencing data of the three clinical NPA samples, it was observed that neither of these samples has identical DI formation pattern in any of the polymerase genes, apart from a few conserved DI patterns that was identified in multiple samples. Given that the current understanding in the formation of influenza DI-RNA is limited, the exact mechanism leading to the diversity of DI-RNA species production is still unknown. It could be possible that either the polymerase complex initiates the generation of DI-RNA in a random manner or the molecular difference of the sequence in the internal zgenes of these patients could give rise to this observation. With the feasibility to identify exact DI species present with influenza infection in cultured cell system, the molecular mechanism of DI-RNA generation could be elucidated with bioinformatics analysis of SMRT-sequenced In addition to the identification of multiple DI-RNA species in SMRT sequencing, similar or identical sequences are shown around the breakpoint region and the other side of deleted region (previously described as overlapping sequences ), which Robust expression of DI-RNA was observed in the H7N9-infected clinical specimens, cell culture and mice. Recent study suggested that the reduction of DI-RNA production in H1N1 viruses led to low antiviral response induction and increased viral pathogenesis . UnfortuIn conclusion, our data demonstrates the existence of DI-RNAs in clinical specimens and cultured cells and mice model during influenza A (H7N9) virus infection. We also show that SMRT sequencing is a promising alternative sequencing technique which provides comprehensive genetic information and relative abundance of multiple DI-RNA species. Using the third-generation sequencing, we identify multiple abundant DI-RNA species in different clinical specimens and their overlapping sequences at the breakpoint regions. Increased DI-RNA synthesis is observed in H7N9-infected NHBE cells compared to the reported H1N1 (WSN) strain that generates DI-RNAs. All the studies mentioned above revealed the overwhelming synthesis of DI-RNAs during infection of H7N9 influenza A virus.The raw data of the SMRT sequencing reads were submitted to Genbank under Genbank accession number SRP126530.


Penicillium digitatum, has an unresolved initial infection mechanism. Understanding the infection mechanism leads to the development of potential controls and preventive measures against the disease. The present study aimed to delineate the infection mechanism by investigating spore germination, changes of organic molecules and enzyme activity, and differential expression of genes in the P. digitatum infection. P. digitatum spore germination was observed by a pathology section scanner and it was found that in vivo germination was 3 h behind the in vitro germination. In addition, cell wall degrading enzymes and soluble sugar and titratable acid content during the infection process measured dynamically. The level of pectinase reached its maximum of 6067 U/g before 48 hpi, while cellulase increased rapidly after 48 hpi. The soluble sugar and organic acid content increased considerably with the progression of the infection. The transcriptomic profile of P. digitatum before and after infection was analyzed by RNA-seq. The genes related to cell wall degrading enzymes were significantly up-regulated and annotated to participate in two major carbon source synthesis pathways. The study delineated the initial infection mechanism of P. digitatum which eventually opened the gate way for the development of new control strategies in the future.Green mold disease, a common citrus post-harvest disease caused by  P. digitatum, is the most serious infection of citrus [P. digitatum were reported to have the ability to become resistant against common fungicides, so there is a growing demand for safe and effective alternative control methods [Citrus fruits grow on flowering trees and shrubs, and are known as one of the four big fruits of the world. Citrus is delicious in taste and is a rich source of ascorbic acid and dietary fiber . Howeverf citrus . Currentf citrus . Some mo methods .P. digitatum was reported in high diversified fruits, but their parasitic ability is weak. In most cases the pathogen infects the fruit and lurk on the surface, but does not affect the fruit quality. P. digitatum invade into the tissue only when the tissues of the fruit are bruised or wounded [Penicillium strains are between 23 °C and 25 °C. The most notable Penicillium spps. like P. italicum grow optimally at 23 °C, and the optimum growth temperature of P. digitatum is 25 °C which is sparingly greater [Pathogen invasion into plant tissue or cells is a complex process in which the pathogens need to break several barriers like plant epidermal tissue, cell wall and the immune system of plant cells . Green m wounded . After s greater .1) and beta-glucosidase (β-G) [P. digitatum of citrus.Though the pathogens enter into the host and have suitable environment for their growth and proliferation, the cell wall of the host acts as a primary defense barrier to reduce pathogen attacks . The basse (β-G) . SimilarColletotrichum fragariae in strawberry leaves with fluorescent markers. In addition, Yuan et al. [Fusarium oxysporum infected strawberries by scanning electron microscopy, and further explored the interaction mechanism between pathogen and host. However, no article has been found on the microscopic observation of the postharvest infection of P. digitatum on citrus. Microscopic observation can bring an intuitive understanding of the infection process of pathogens, and correlated with findings from some other infection mechanisms of the pathogen, also identify key time points. However, the infection mechanism of P. digitatum in citrus fruits is not fully understood, therefore, it was of great significance that we studied the infection process of P. digitatum and the mechanisms involved at the physiological and molecular levels. The exploration of the pathogen infection process into plant tissue through microscopic observations can give a deeper understanding of pathogen infection mechanisms. A few examples of such observations were reported by Zhao et al.  where thn et al.  studied P. digitatum in citrus wound tissues was investigated by sectioning the tissues pathology section scanner. (2) The activity of the main cell wall degrading enzymes, soluble sugar and titratable acid contents of citrus fruits during the process of infection was measured dynamically. (3) The key time of early infection process of P. digitatum was obtained based on the results of above-mentioned experiments. The transcriptomic profile of P. digitatum before and after the infection was analyzed by RNA-seq. Subsequently, we focused on the expression of genes related to cell wall degrading enzymes and their metabolic pathways. This study provides a good knowledge of elucidation about infection mechanism of P. digitatum on postharvest citrus during initial infection process.In the present study: (1) The early infection process of P. digitatum by sequencing the 5.8S internal transcribed spacer (ITS), beta-tublin (BenA) and Calmodulin (CaM) DNA regions and sequence similarity search in NCBI GenBank [P. digitatum culture in sterile saline and were adjusted to an appropriate concentration (1 × 106 spores/mL) using a hemocytometer [The pathogen was isolated from natural rotten citrus and identified as  GenBank . Spore sytometer .Citrus reticulata Blanco) of similar color and maturity stage that were free of any visible mechanical damage were selected from an orchard, and further used for experiments [Citrus fruits . The decay incidence and lesion diameter of the treated citrus fruits were recorded every 12 h [Three identical wounds (3 mm width × 5 mm deep) were created on the equator of the citrus fruits after washing and sterilizing in 1% sodium hypochlorite. 30 µL of the ery 12 h . Three rP. digitatum was spread on the surface of the PDA plates and cultured at 25 °C, and 95% relative humidity. Tissues of samples (treated portions of the citrus fruits) were excised with a scalpel and stained with carbonic acid lactate cotton blue stain for 5 min. Neutral glue was used to fix it to a slide. After air-drying, the excess neutral glue on the edge of the cover slide was cleaned with xylene and the infection process was observed at different time intervals  with a panoramic MIDI slide scanner. There were three replicates per treatment, and the entire experiment was performed three times.An amount of 2.5 µL of the spore suspensions of Preparation and observation of citrus wounds were done as described above in The method used in conducting this experiment was a slightly modified version of that described by Mendes .P. digitatum, a 5 g of the peel was taken from the junction of healthy and diseased on 5 citrus wounds with a sterilized scalpel. An amount of 5 mL of the extraction buffer  was added, and ground in a cold mortar to form a homogenate. More of the extraction buffer was added to make up a 20 mL solution. After shaking thoroughly, it was centrifuged at 18353 × g at 4 °C for 20 min and the supernatant used as extract for enzyme assay.To measure the activity of enzymes secreted by The reaction systems of the enzyme β-glucosidase (β-G) was created as follows: 0.5 mL crude enzyme extract was added to 1 mL 50 mmol/L citric acid-sodium citrate buffer (pH 5.0), 1.5 mL 1% cellobiose solution  and incubated at 50 °C for 60 min in water bath. After 60 min, 3 mL of 3,5-dinitrosalicylic acid reagent (DNS) was added to stop the reaction. Then, the mixture was boiled for 5 min and immediately cooled under running water. To measure the activity of the enzyme, distilled water was added until 15 mL and then the absorbance was measured at 540 nm. The enzyme activity unit (U/g) was defined as the amount of enzyme required to produce 1 µg of glucose per hour at 50 °C. Cellobiose solution was added to the control group after the addition of the DNS reagent. The assay preparation procedure for the activity of Exo-1,4-β-glucanase (Cx) was the same as β-G except that the substrate was 1% sodium carboxymethyl cellulose (CMC).The activity of polygalacturonase (PG) was determined as follows: To 0.5 mL of crude enzyme extract, 1 mL of 50 mmol/L sodium citrate buffer (pH 5.0), and 1.5 mL of 1% polygalacturonic acid solution  was added. After incubating at 50 °C for 60 min in water bath, 3 mL of DNS reagent was added to stop the reaction. Then, the mixture was boiled for 5 min and cooled under running water. To measure the activity of the enzyme, distilled water was added until 20 mL and then the absorbance was measured at 540 nm. The enzyme activity unit (U/g) was defined as the amount of enzyme required to produce 1 µg of glucose per hour at 50 °C. Polygalacturonic acid solution was added to the control group after the addition of the DNS reagent.The preparation of the reaction systems for the determination of pectin methylgalacturonase (PMG) activity was similar to that of PG except that the substrate was 1% pectin. Each treatment was done in three replicates and the entire experiment was repeated three times.The experiment was done by slightly modified version of the method described by Zeng et al. .g for 20 min at 4 °C after 1 h at room temperature. The supernatant was taken at a constant volume of 100 mL. After 30 min of boiling in water bath, it was cooled and filtered.A total of 5 g of tissue with peel was taken from five citrus wounds and ground with 10 mL distilled water. The homogenate was transferred into a centrifuge tube and centrifuged at 4000× To determine the titratable acid contents of the treated samples, 1% phenolphthalein indicator was added to 10 mL of the above extract and titrated against a calibrated sodium hydroxide solution. Distilled water was used instead of the filtrate/extract for titration in the control group.To quantify the soluble sugar contents of the sample, 10 mL of the above extract was taken and diluted to 50 mL. 0.5 mL of anthrone reagent and 5 mL of concentrated sulfuric acid was added into 2 mL of the diluted extract in a test tube. Then the absorbance was measured at 630 nm. Distilled water was used instead of the filtrate/diluted extract for the soluble sugar measurement for the control group.P. digitatum inoculation was done as described above . RNA purity, concentration and integrity were determined with reference to Hsu et al. .Prior to using mRNA as a template, the eukaryotic mRNA was enriched with magnetic beads with Oligo (dT), the fragmentation buffer was used to randomly break the mRNA. Secondly, the first cDNA strand was synthesized with random hexamers, then a second cDNA strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. The purified double-stranded cDNA was repaired with A-tailed and ligated to the sequencing linker, and fragment size selection was performed using AMPure XP beads. Finally, a cDNA library was obtained by PCR enrichment. After the library was constructed, preliminary quantification was performed using Qubit 2.0. The library was diluted, and then Agilent 2100 was used to detect the insert size of the library. After the insert was found to be in accordance with expectations, the effective concentration of the library was accurately quantified using RT-qPCR. After the library was tested, different libraries were pooled to the flow cell according to the effective concentration and the target data volume. The cBOT was then clustered and sequenced using the Illumina high-throughput sequencing platform (HiSeq/MiSeq) . The traRaw data was filtered to obtain high quality and clean data. The clean data was sequence aligned with the specified reference genome (GCF_000315645.1_PdigPd1) to obtain a mapped data. DESeq2 was used to analyze the differential expression level of each gene in two samples (|log2(Fold Change) | ≥ 1 and false discovery rate (FDR) < 0.05 were used as screening criteria). The significance of the p-value of the original hypothesis test was corrected using the accepted Benjamini–Hochberg correction method. p < 0.05. Results were presented in tables and figures.The data were analyzed for significant differences by analysis of variance (ANOVA) with the Minitab statistical package (version 17), using the Tukey test at P. digitatum at 25 °C recorded a significant increase in rot at 48 h post inoculation (hpi) compared to the control sample, and the decay incidence was observed increasing rapidly and reached 100% at 60 hpi. At this time, the diameter of the decay was about 26.02 mm. An extension in culture time, revealed a rapid expansion of the diameter of the decay. The whole fruit was decayed (69.78 mm) at 108 hpi, unlike the control group which was not showed any decay , 36 hpi (3463 U/g) and 72 hpi (3546 U/g), which were 1.36 times, 1.57 times and 1.61 times that of the control group, respectively. There were no significant changes in activity of samples in the control group  on day four, after which it declined slowly  before infection were 24285615 and 398363318, and the GC Content were 54.46% and 54.51%, respectively. However, clean reads of P. digitatum (44 h) after inoculation into the wounds of citrus fruits were 32,810,982 and 51,322,479, and the GC Contents were 54.39% and 54.47%, respectively. In terms of statistical evaluation of base mass values, the Q30 base percentage of the four groups of samples reached above 94.39%.A total of 44.48 Gb clean data were obtained after transcriptome sequencing. The Clean Reads of In addition, the differentially expressed genes (DEGs) were functionally annotated with databases such as gene ontology (GO), SwissProt and Kyoto Encyclopedia of Genes and Genomes (KEGG). From the results we have found that some genes were related to CWDEs. Out of the lot (≥70 genes), that were annotated to participate in the synthesis of CWDEs in the SwissProt, 12 genes were annotated as extracellular regions in GO cellular component . Three oPdCELB, PDIP_47720), , (eglB(PDIP_65210), ,  and  were involved in starch and sucrose metabolism (ko00500) ,  and  were also found to be involved in pentose and glucuronate interconversions (ko00040)  A. They wko00040) B. They wP. digitatum is considered a most alarming disease of citrus [P. digitatum in citrus fruits was revealed, combined with the studies that proved the changes/activities of cell wall degrading enzymes in the infection mechanisms of P. digitatum in citrus. Moreover, RNA-seq was performed on P. digitatum at key times to ascertain the role of cell wall degrading enzymes at the molecular level.Postharvest green mold decay caused by f citrus . In recef citrus . On the P. digitatum lacks the capacity to break through the cuticle on the stratum corneum of citrus surface, so the necessary nutrients for growth must be obtained from the wound. The water-stained soft rot appeared between 36 and 48 h at 25 °C after inoculation of P. digitatum spores in the wounds of citrus. The lesion incidence was more than 90% at 48 h and this observation was in agreement with the findings of Vilanova et al., [P. digitatum spores begun to infect citrus wounds within a short time and begun to colonize. Although P. digitatum is the main pathogen of postharvest diseases of citrus, its reproductive cycle is still unclear [P. digitatum spores in vitro and in vivo. et al., . Results unclear . TherefoP. digitatum spores had been activated and swelled at 6 hpi in vitro, after which it started to form germ tubes. The development of the germ tube was completed between 6 and 9 hpi, and then the germ tube continued to elongate to form a thick branched mycelium before 28 hpi. The phialide structure formed at the end of mycelium 36 hpi, then new conidia were formed on phialide and dropped off at 44 hpi. The results of in vivo growth were consistent with the in vitro studies, however, the interesting part is that the growth of the spores in vivo showed a difference of about 3 h as compared to that in the in vitro growth. It is probably because the spores of P. digitatum could directly acquire the carbon source necessary for growth on the PDA, while there is the need to secrete a series of cell wall degrading enzymes from spores to obtain nutrition in citrus fruit wounds [The pathogenic fungus became activated after contact with the host, and the spores gradually expanded after absorbing nutrients . SubsequP. digitatum first secreted a series of pectinases to degrade the pectin in the intercellular layer to produce the carbon source necessarily for its growth after invading citrus fruit wounds  by pectinase, thereby obtaining a large number of nutrients for its rapid proliferation [P. digitatum  and after 44 hpi on citrus wounds, the results revealed that expression of genes related to pectinase and cellulase synthesis increased significantly after the spores of P. digitatum were inoculated into citrus wounds  was up-regulated 21.41 times. It is a cell wall degrading enzyme based on hemicelluloses [P. digitatum infection process of citrus. Furthermore, cellulase-related genes were annotated to participate in starch and sucrose metabolism (ko00500)   A, which ko00040) B. The meko00040) B, which rganisms . Starch P. digitatum in wound tissues of citrus fruits during the initial infection process by microscopic observation, enzyme activity, physicochemical measurement and comparative transcriptome analysis. In addition, there is still a huge number of data in the RNA-seq that need to be analyzed. There are more DEGs available, which play important role in fungal development and pathogenicity, cause the formation of invasive growth and variation, and produce toxins related to fungal infection of plants which we are interested in. Furthermore, more bioinformatics analysis and functional validation are needed for these genes. Furthermore, considerations on revealing the mechanism of postharvest P. digitatum infection in citrus fruits from these analyses will be done, to control the infection. In conclusion, the study elucidated the infection mechanism of


Observational studies and randomised controlled studies suggest that vitamin D plays a role in the prevention of acute respiratory tract infection (ARTI); however, findings are inconsistent and the optimal serum 25-hydroxyvitamin D (25(OH)D) concentration remains unclear. To review the link between 25(OH)D concentration and ARTI, we searched PubMed and EMBASE databases to identify observational studies reporting the association between 25(OH)D concentration and risk or severity of ARTI. We used random-effects meta-analysis to pool findings across studies. Twenty-four studies were included in the review, 14 were included in the meta-analysis of ARTI risk and five in the meta-analysis of severity. Serum 25(OH)D concentration was inversely associated with risk and severity of ARTI; pooled odds ratios  were 1.83 (1.42–2.37) and 2.46 (1.65–3.66), respectively, comparing the lowest with the highest 25(OH)D category. For each 10 nmol/L decrease in 25(OH)D concentration, the odds of ARTI increased by 1.02 (0.97–1.07). This was a non-linear trend, with the sharpest increase in risk of ARTI occurring at 25(OH)D concentration < 37.5 nmol/L. In conclusion, there is an inverse non-linear association between 25(OH)D concentration and ARTI. Acute respiratory tract infection (ARTI) is very common, with most people experiencing at least one episode of ARTI each year . ARTI inVitamin D is critical for skeletal health and may play a role in other health outcomes, including infection. Vitamin D status is estimated by measuring the serum concentration of 25-hydroxyvitamin D (25(OH)D); this varies seasonally and the lowest concentration in the winter/spring months coincides with the highest ARTI incidence, suggesting a link between vitamin D and ARTI . This isMany studies have investigated the link between 25(OH)D serum concentration and ARTI and the effect of vitamin D supplementation on ARTI. Two recent meta-analyses of observational studies considered associations between 25(OH)D concentration and ARTI incidence in children ,10. One Results from RCTs investigating the effect of vitamin D supplementation on ARTI are inconsistent. Two meta-analyses found a significant benefit of vitamin D supplementation on ARTI ,18 whileThe study protocol was registered with the PROSPERO International Prospective Register of Systematic Reviews prior to commencement .PubMed and EMBASE databases were searched from their inception until 12th June 2019. Keywords were chosen from the Medical Subject Headings (MeSH) terms in PubMed and explosion (exp) of EMTREE terms in EMBASE. Essentially, we searched for the terms “vitamin d” or “25-hydroxyvitamin D” or “25OHD” or “25(OH)D” or “hypovitaminosis D” in combination with “respiratory tract infection*” or “respiratory infectio*” or “respiratory diseas*” or “pneumonia” or “influenza” or “bronchiolitis” or “common cold”. The complete search strategies are shown in the All titles, abstracts and full text were independently screened by two investigators, Hai Pham and Aninda Rahman. Any discrepancies were resolved through consultation with Rachel Neale and Mary Waterhouse.Inclusion criteria: The eligibility criteria were: (1) observational studies including cohort, case-control and cross-sectional studies; (2) published in English; (3) full text available; (4) reported the association between circulating 25(OH)D concentration and ARTI risk or severity; (5) the target population was healthy adults or adolescents aged 12 years and older.Exclusion criteria: Studies investigating the link between 25(OH)D concentration and tuberculosis or chronic lung conditions such as chronic obstructive pulmonary disease and asthma were excluded.Primary outcome: The primary outcome was the risk of ARTI, defined as an acute infection of the respiratory tract in either the lower or upper airway or with the location not specified. ARTI was either self-reported via surveys or symptom diaries, or clinically confirmed with or without evidence from X-rays or laboratory tests.Secondary outcome: The secondary outcome was the severity of ARTI, defined according to the duration of the illness, hospitalisation, admission to an intensive care unit, symptom severity score or index, or mortality.We used the customised Newcastle-Ottawa scale (NOS) to assess the quality of each study . The NOSA measure of association between 25(OH)D concentration and ARTI risk or severity (odds ratio (OR), relative risk (RR), hazard ratio (HR), mean difference (MD)) and their 95% confidence interval (CI) or standard deviation (SD); orSufficient data to derive two by two tables of ARTI risk, comparing the lowest versus the highest 25(OH)D category.We used STATA 13  and SAS 9.4  for statistical analyses. We included studies in the meta-analysis that reported either:We included the estimate from the most fully adjusted model for each study in the meta-analysis comparing the risk of ARTI in the lowest versus highest 25(OH)D category. When a study reported results using different 25(OH)D thresholds, we used 50 nmol/L as it was the most commonly used threshold. Two studies reported HRs as the measure of association between 25(OH)D concentration and risk of ARTI ; we inclWe used a method described by Greenland and Longnecker to estimate trends across categories of exposure to calculate the effect of each 10 nmol/L decrease in 25(OH)D concentration on the risk of ARTI ,28. ThisFor the overall analysis of severity, we defined a severe ARTI to be one that had a moderate-to-high severity score or resulted in death (no studies based on other severity outcomes were included). We also performed a separate meta-analysis to assess the association between 25(OH)D concentration (lowest versus highest category) and ARTI mortality.2 statistics. We conducted meta-analyses comparing the risk of ARTI in the highest versus the lowest 25(OH)D category stratified by: (i) infection location if reported (URTI or LRTI); (ii) outcome measurement methods ; (iii) mean 25(OH)D concentration in the study population ; and (iv) factors considered as confounders (fully adjusted model or crude/non-fully adjusted model) to explore potential sources of heterogeneity. We used funnel plots and the Egger test to test for publication bias.Heterogeneity across studies was assessed using IThe characteristics of the included studies are summarised in n = 6). ARTI severity was measured using a severity index (n = 6) such as CURB or CURB-65 , pulmonary severity score (PSI), or symptom severity score (SSC); duration of the illness (n = 10); admission to hospital or intensive care units (n = 4); or mortality (n = 6). All 24 studies had a moderate-to-high quality score (ARTI risk was measured by (i) self-report via survey questionnaire or symptom diaries (n = 10), or (ii) clinical diagnosis with or without confirmation from X-ray or a laboratory test  and 10 studies were included in the trend analysis . Five studies reported risk of ARTI in at least three categories of 25(OH)D concentration and the number of cases by exposure category; these were included in the evaluation of whether there is a non-linear relationship between ARTI risk and 25(OH)D concentration .We included 14 of the 16 studies that assessed the association between 25(OH)D concentration and risk of ARTI in the meta-analysis comparing the risk of ARTI in the lowest versus the highest 25(OH)D category   (p for non-linearity = 0.029) with inflexion points at 60 nmol/L and 37.5 nmol/L. The steepest increased risk occurred below 37.5 nmol/L (There was a significantly higher risk of ARTI in the lowest compared with the highest 25(OH)D category  . The poo< 0.001) . There w5 nmol/L . AlthougN participants = 1495) were included in the meta-analysis of the odds of severe ARTI or mortality combined, comparing the highest and the lowest 25(OH)D category and four studies (N participants = 1422) were included in the mortality meta-analysis. The pooled ORs for severity/mortality combined and mortality separately were 2.46  and 3.00 , respectively ; therefore, we conducted stratified meta-analyses to explore potential sources of heterogeneity  (We found evidence of significant heterogeneity (Iogeneity A–D. The ogeneity A. Similaogeneity C. There < 0.001) D.p = 0.024) (The funnel plot shows evidence of significant publication bias (= 0.024) , suggestIn this systematic review and meta-analysis, we observed significant associations between 25(OH)D concentration and both risk and severity of ARTI, but with significant heterogeneity and evidence of publication bias. There was a non-linear association between 25(OH)D concentration and risk of ARTI, with evidence of a more marked increased risk for 25(OH)D concentration below 37.5 nmol/L.n = 44,301) reported an increased risk of ARTI in those with 25(OH)D < 50 nmol/l compared with those ≥ 50 nmol/L. The odds ratio was higher than we found (2.63 vs. 1.83); their analysis included children as well as adults and they did not present an estimate for adults and adolescents only [n = 550) [Findings from our meta-analysis of ARTI risk, which includes the largest number of participants, are consistent with results from previous reports. A meta-analysis published in 2015 as an abstract (nts only . A systents only . The linn = 550) .This is the first study to our knowledge which performed a meta-analysis of the association between 25(OH)D concentration and severity of ARTI. We found a stronger association between 25(OH)D concentration and severity of ARTI than with risk of ARTI (OR 2.46 vs. 1.83). The result accords with findings from a systematic review that highlighted the potential link between vitamin D deficiency and severe LRTI in children, but no meta-analysis was included . It is dResults from RCTs are inconsistent. Some trials found that vitamin D supplementation reduced the risk of ARTI ,49,50 whWe observed high heterogeneity, and this mostly persisted in subgroup analyses. Despite this, the direction of effect was consistent; lower 25(OH)D concentration was associated with increased risk or severity of ARTI. There are indications of publication bias, indicating that small studies that showed no significant association were either not identified or not published. Publication bias has also been observed in meta-analyses of RCTs ,22. It iOur study is the largest to date and to the best of our knowledge, is the first to include a meta-analysis of the association between 25(OH)D concentration and severity of ARTI. Our findings suggest an important role of vitamin D in prevention of ARTI risk and severity, particularly in people with low 25(OH)D concentration. However, it is challenging to identify an optimal 25(OH)D concentration or a concentration below which supplementation would be of benefit due to the lack of consistency in both 25(OH)D assays and reporting across studies. It is important to improve consistency of reporting, as well as assays, to enable the field to move forward.


The preferred product characteristics , in addition to safety and efficacy, are quintessential requirements for any successful therapeutic. Messenger RNA vaccines constitute a relatively new alternative to traditional vaccine development platforms, and thus there is less clarity regarding the criteria needed to ensure regulatory compliance and acceptance. Generally, to identify the ideal product characteristics, a series of assays needs to be developed, qualified and ultimately validated to determine the integrity, purity, stability, and reproducibility of a vaccine target. Here, using the available literature, we provide a summary of the array of biophysical and biochemical assays currently used in the field to characterize mRNA vaccine antigen candidates. Moreover, we review various in vitro functional cell-based assays that have been employed to facilitate the early assessment of the biological activity of these molecules, including the predictive immune response triggered in the host cell. Messenger RNA vaccines can be produced rapidly and at large scale, and thus will particularly benefit from well-defined and well-characterized assays ultimately to be used for in-process, release and stability-indications, which will allow equally rapid screening of immunogenicity, efficacy, and safety without the need to conduct often lengthy and costly in vivo experiments. Over the last decade, messenger RNA (mRNA) has emerged as a new platform technology for the development of vaccines not just for anti-cancer therapeutics but also against infectious diseases. As reviewed in the accompanying manuscript by Versteeg et al., there is now even progress in the challenging development of mRNA vaccines against parasitic infections where a low-cost and high-throughput vaccine development platform is urgently needed .Messenger RNA vaccines are composed of one or more nucleic acids encoding for a single or multiple vaccine antigen candidates, formulated in a delivery vehicle and then directly delivered into the host cell. These delivery vehicles include lipid- and protein-based molecules, as well as various types of polymeric scaffolding ,6,7,8,9.The mRNAs’ biochemical and biophysical properties promise to allow for simple multiplexing of vaccine antigens without the formulation complications that can be encountered with protein-based vaccines, or the potential risks from attenuated whole-cell vaccines. Moreover, in contrast to DNA vaccines, RNA vaccines exert their function directly in the cytoplasm, depending on neither transport mechanisms nor on the cellular transcription machinery. The fact that RNA cannot integrate into the host genome and that it has a short half-life that limits its persistence in the body contributes to a favorable prediction of its safety profile. A longer half-life can be achieved with self-amplifying mRNA (SAM); SAM vaccines are engineered into the backbone of an RNA virus genome, allowing amplification of the antigen-coding region resulting in higher levels of expression. Since the virus’ structural genes are absent in SAM constructs, there is no associated risk of infection ,11.As reviewed elsewhere ,6, mRNA Long-established for other biologics, an array of quality control measures  will need to be developed to identify the preferred product characteristics during in-process, release and stability evaluation of the vaccine targets and to ensure a reproducible safe product for pre-clinical and clinical use .The success of RNA vaccines as defined by safety, immunogenicity, and efficacy will depend primarily on the quality of the transcript ,12,13,14In this review, we provide a summary of the preferred quality control assays currently used to characterize mRNA vaccine antigen candidates at the biophysical, biochemical, and functional level. Even though most of these assays are widely used and well-established in the research laboratory, further optimization, qualification, and eventual validation will be needed to transition them for use during manufacturing campaigns. In fact, so far, no regulatory guidelines specific to mRNA vaccines quality control have been provided , althougMessenger RNA vaccines can be designed with great customization. For example, rare codons can be replaced to increase translation, and undesired secondary structures can be removed to avoid an undesired innate immune response . DNA temFollowing production, RNA transcripts are digested with DNAse I to degrade DNA templates, and then purified to remove DNA degradants, reaction components, and other contaminants. Purification method include commerical spin columns, LiCl precipitation, phenol-chloroform extraction, and chromatography ,24,25.The most straightforward method to characterize RNA transcripts prima facie is UV spectroscopy ,27. For Care should be taken to measure absorbances against appropriate blanks, since many common biochemical reagents absorb UV light strongly, especially at shorter wavelengths, including buffers such as N-(2-acetamido)iminodiacetic acid. Indeed, non-specificity is a critical issue in UV spectroscopy, as for example, RNA degradation is undetectable by UV spectroscopy. Moreover, residual plasmids and nucleotides, if present, will also absorb UV light at 260 nm. DNAse I treatment is regularly employed to remove deoxyribonucleic acids from RNA preparation, yet the efficacy of the reaction needs to be confirmed. Furthermore, residual enzymes from transcription reactions have absorbance maxima at 280 nm, and other potential contaminants such as guanidine thiocyanate, a common component in commercial nucleic acid purification kits have absorption maxima in the UV range as well. Accordingly, A260/A280 and A260/A230 ratios are often superior indicators of purity, with highly-quality RNA producing ratios of 1.8–2.2 and >1.7, respectively [The fluorescence of certain dyes, when bound to nucleic acids , can be Following separation by molecular mass on agarose or acrylamide gels, nucleic acids can be visualized using fluorescent dyes such as ethidium bromide, SYBR Green or SYBR Gold  ,33,34, aWhile gel-based methods are less sensitive, some specificity is achieved since the target RNA can potentially be separated from residual plasmid DNA or degradants. Nevertheless, contaminating proteins would still have to be quantified separately by other means. Importantly, gel-based methods can be used to measure RNA size and integrity. For instance, the 2100 Bioanalyzer outputs an RNA Integrity Number between 0 and 10, 10 representing RNA of high quality .Although not typically employed for this purpose, reverse transcriptase qPCR can also be used to quantify or characterize RNA transcripts produced in vitro. Messenger RNA transcripts are first reverse-transcribed to cDNA and then quantified by standard qPCR using DNA-binding fluorescent dyes or fluorescently labeled DNA probes or primers. As before, standards of known concentration can be assayed at the same time to measure the amount of RNA in each sample. If suitable primers can be designed, then the target RNA can also be directly quantified, as well as any residual plasmid. Primers that generate amplicons of various lengths can be used to determine RNA integrity since larger amplicons become consistently more difficult to amplify as RNA degrades. RT-qPCR is fast, high-throughput, and extremely sensitive, although it requires advanced training, capital equipment, and expensive reagents and consumables.As double-stranded RNA is strongly immunogenic, its detection in and removal from RNA vaccines is critical, especially if RNA transcripts were produced in vitro . These s32P) GTP during in vitro transcription [Capping at the 5′ end is a hallmark of eukaryotic and viral messenger RNA. Since capping affects stability and translation, proper quality control should include the quantitative assessment of capped and uncapped transcripts. In one method, a biotinylated tag is annealed to the 5′ end of the transcript, which is then cleaved via DNA 3′ end-directed cleavage by RNAse H. Subsequently, the hybrid fragment is isolated on streptavidin-coated magnetic beads, dissociated, and the untagged strand is analyzed by liquid chromatography-mass spectrometry (LC-MS). This method was reported to detect 0.5–25% uncapped RNA in 100 pmol of capped RNA . If the cription , which, cription . This mecription .Messenger RNA transcripts can be quantified or characterized by chromatography, including size-exclusion chromatography ,46,47, aAlthough naked RNA transcripts can be physically delivered into cells by electroporation, they are, instead, often formulated with excipients that confer additional functional properties. For example, most delivery vehicles are expected or designed to protect against RNAses, and such protection is evaluated by electrophoretic mobility shift assay . Transcr2+, K+, etc.). We note that although commercial systems are expensive, individual reactions can be scaled down to as low as 1 µL in order to drastically reduce costs when screening multiple transcripts, although care should be taken to prevent evaporation, such as by using a heated a lid.To ensure that the mRNA transcript is in-frame and contains all elements necessary for translation, it can be translated in vitro using cell-free extracts ,50. AlthProducts from in vitro translation can then be visualized using standard Western blot if appropriate antibodies are available. If necessary, tags can be engineered into the transcript to enable immunodetection. Alternatively, the in vitro translation reaction can be supplemented with fluorescently labeled amino acids  which arThe next step for a complete evaluation of mRNA vaccine antigen candidates is to test the transfection of the molecules into the target cell and confirm their suitability for effective translation. In mRNA vaccine development this in vitro testing offers advantages over in vivo studies; a larger number of variables can be investigated and controlled in each experiment and the potential toxicity of specific molecules can be detected in a specific, safe and efficient way. This can be particularly useful when multiple vaccine candidates are being screened before entering the essential in vivo testing for validation.Transfection rates can be best measured by Flow Cytometry using fluorescently labeled nucleotides, incorporated into the transcription reaction. Single-cell arrays are also available to rapidly assess transfection in support of parallel screening of multiple mRNA candidates ,53. Tran6 RLUs (Relative Light Units), in keratinocytes they only observed 2.5 × 106 RLUs. The authors further observed that the cell line background influenced the impact of certain nucleoside modifications on translation efficiency; m5C/m1ψ had the biggest effect in all cell lines except C2C12, where actually m1ψ yielded the biggest benefit, further emphasizing the importance of considering what cell line is used in a particular experiment.When mRNA molecules are then first examined in cells, the context of the test system needs to be considered, as some cell lines are easier to transfect than others and translation efficiencies may also differ based on the host system. For example, Andries et al. evaluated modified nucleosides such as pseudouridine, N1-methylpseudouridine (m1ψ), 5-methylcytosine/pseudouridine and 5-methylcytosine/N1-methylpseudouridine (m5C/m1ψ) for their effect on transfection rates using self-reporting luciferase encoding mRNA. With this approach, they not only found generally increased transfection rates with m5C/m1ψ but, equally important, they also identified a more than 20-fold different luciferase activity depending on the target cell line . While iTo date the most commonly used cell type to test mRNA vaccines are dendritic cells (DCs), antigen-presenting cells (APC) that can stimulate  naïve T cells, resulting in cell-mediated immunity. Upon pathogen recognition, the innate and adaptive immune responses can upregulate components of the Major Histocompatibility Complex (MHC) molecules as well as co-stimulatory receptors. In addition, DCs can promote self-tolerance by secreting tolerogenic cytokines that induce the differentiation of regulatory T cells. As a result, there is considerable interest in DCs as potential therapeutic targets . The cha+ or CD8+ T cells) and have the ability to migrate to the lymph nodes to present antigens to T cells. In the lymphoid organs and other tissues, the conventional tissue-resident DCs (cDC) act as sentinels for antigen capture and presentation in situ. Other inflammatory DCs may differentiate from blood-derived monocytes and infiltrate secondary organs and tissue during infection as part of the inflammatory response [There are two different stages in the life cycle of DCs: Immature DCs (imDC), found in the periphery where they patrol pathogens, apoptotic cells, and antigens from the outside and process them with great efficiency. However, their ability to present antigens and stimulate T cells is limited. They are also characterized by low cytokine production and low expression of MHC molecules. Mature DCs (mDCs), though, are characterized by a high expression of MHC components, as well as co-stimulatory and adhesion molecules. Furthermore, mDCs are efficient in antigen-presentation to T cells  or peripheral blood mononuclear cells (PBMCs), immortalized cell lines save both time and costs  as they Transfection efficiency of mRNA construct has therefore primarily been tested in DC2.4 cells. For example, this immortalized cell line was used to test mRNA delivery using self-assembled cationic nanomicelles. Uptake was evaluated using fluorescently labeled mRNA in conjunction with various delivery platforms and quantified using flow cytometry. The expression of the mRNA was verified using EGFP (enhanced Green Fluorescent Protein) encoding mRNA, and the resulting protein was again quantified by flow cytometry. Overall, the authors were able to use this approach to demonstrate the benefit of their nanoparticles for the translation of the target RNA . To furtIn another example of the use of DC2.4 cells to quality control therapeutic mRNA candidates, Haifa Shen’s laboratory is studying cellular uptake of their anti-cancer therapeutics. Efficient internalization was initially tested using GFP (Green Fluorescent Protein) as a self-reporting marker, combined with Flow Cytometry to quantify the number of GFP-positive cells. Without having to go into animals, the authors were further able to test the uptake of fluorescently labeled mRNA in the presence of various uptake inhibitors, to better understand the endocytic pathway of how their particular mRNA vaccine enters the dendritic cells .Aside from Flow Cytometry, fluorescence Microscopy has proven to be a valuable tool to monitor transfection and the time course of antigen mRNA expression and to evaluate different delivery systems in vitro ,64,65,66Other studies to understand the innate immune response produced by mRNA constructs have been conducted using BMDCs. When DCs from BALB/c mice were transfected with mRNA (Luciferase and EGFP), with and without modified nucleosides, a reduction in the innate immune response was associated with the modification likely also responsible for an increase in translation. The same study used mRNA purified by HPLC, and conclude that the purification increased the translation while also controlling the innate immune response . StudiesAlternative types of cells have also been used to evaluate and characterize mRNA vaccine candidates against specific pathogens before in vivo evaluation. The PR8HA antigen, for instance, was tested in HeLa cells as an mRNA vaccine candidate against Influenza A. Translation efficiency invitro of the PR8HA mRNA was measured by flow cytometry, as well as intracellular (99.6%) and surface (42.7%) staining. After the high translation efficiencies observed in the cell culture model, testing was moved to the mouse model, where 100% survival upon lethal challenge was observed . In 2013Streptococcus pyogenes (GAS) and Group B Streptococcus agalactiae (GBS).In another example, mRNA vaccines against the rabies virus encode the surface glycoprotein RABV-G. Before being used in animals, the translation of the RABV-G was tested in HeLa cells by flow cytometry, where 99.6% of the cells were positive for the antigen. Subsequently, the survival of challenged and vaccinated mice was 100% against lethal intracerebral rabies challenge infection . For theMessenger RNA as a novel vaccine platform holds great promise, but, as all biologics, it will require careful characterization in order to ensure quality, safety, and efficacy. We have tried to compile an array of published biophysical and biochemical tools as well as cellular immunology tools that are necessary to evaluate the quality characteristics of vaccine antigen candidates before advancing them into more costly pre-clinical testing. With this toolbox of assays in place, mRNA vaccine antigen candidates can swiftly be advanced towards in-vivo testing. It is noted though that the efficacy of an mRNA in vivo will likely strongly depend on its formulation and the delivery platform. Even at the level of cellular testing, multiple approaches are still being investigated  and whil