Rickettsia species remain poorly understood. Here, we report on the identification of both novel and well-known bacterial sRNAs in Rickettsia prowazekii, known to cause epidemic typhus in humans. RNA sequencing of human microvascular endothelial cells (HMECs), the preferred targets during human rickettsioses, infected with R. prowazekii revealed the presence of 35 trans-acting and 23 cis-acting sRNAs, respectively. Of these, expression of two trans-acting (Rp_sR17 and Rp_sR60) and one cis-acting (Rp_sR47) novel sRNAs and four well-characterized bacterial sRNAs  was further confirmed by Northern blot or RT-PCR analyses. The transcriptional start sites of five novel rickettsial sRNAs and 6S RNA were next determined using 5′ RLM-RACE yielding evidence for their independent biogenesis in R. prowazekii. Finally, computational approaches were employed to determine the secondary structures and potential mRNA targets of novel sRNAs. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis.Emerging evidence implicates a critically important role for bacterial small RNAs (sRNAs) as post-transcriptional regulators of physiology, metabolism, stress/adaptive responses, and virulence, but the roles of sRNAs in pathogenic  A hallmark feature of cis-acting sRNAs, therefore, is nearly perfect nucleotide complementarity to the target genes, unlike only partial nucleotide complementarity displayed by trans-acting sRNAs. Due in part to this partial complementarity, trans-acting sRNAs generally require the involvement of an RNA chaperone to facilitate nucleotide binding  in pathogenic bacteria have garnered immense recent attention due to their ability to control diverse, physiologically important lifecycle processes such as quorum sensing, metabolism, stress responses, and virulence. Typically ranging from 50 to 500 nucleotides in length, sRNAs are heterogeneous in size and structure. Despite being longer in length, these sRNAs are considered to be analogous to eukaryotic sRNAs in the context of certain functional implications. Post-transcriptional sRNA-mediated regulatory mechanisms are broadly categorized into sRNA-protein and sRNA-mRNA interactions. Interactions with the protein-coding transcripts are further categorized into two groups, namely, trans-acting, and cis-acting  and has historically been dubbed as the scourge of armies due to massive outbreaks in the times of wars until World War I. Because of the precedent and possibility of its use as a potential bioweapon, R. prowazekii is also classified as a select agent. In humans, endothelial cells lining the small and medium-sized blood vessels are the primary targets of infection and illness is characterized by progressive endothelial damage leading to widespread vascular dysfunction and enhanced permeability from the intravascular compartment to the interstitium and culminating in generalized vasculitis  of non-coding DNA , epidermal growth factor (10 ng mL−1), hydrocortisone (1 μg mL−1), and 10% heat-inactivated fetal bovine serum and grown at 37°C with 5% CO2 until ~80 to 90% confluency  and plaque assay  were cultured in MCDB131 medium supplemented with L-glutamine  protocol. The RNA samples were treated with DNaseI (Zymo Research) to ensure removal of any contaminating genomic DNA and further processed sequentially through MICROBEnrich (Ambion) and Ribo-Zero (Epicentre) kits to remove interfering eukaryotic mRNAs and ribosomal RNAs, respectively. The enriched RNA preparations thus obtained were quantified using the MultiSkan Go Microplate Spectrophotometer (ThermoScientific) and assessed for the quality control on an Agilent 2100 Bioanalyzer (Agilent Technologies). Two independent cDNA libraries from enriched but non-size selected RNA samples for each experimental condition were then constructed using the TruSeq RNA Sample Prep Kit (Illumina) as per manufacturer's directions. Strand-specific sequencing was carried out on an Illumina HiSeq 1500 instrument at our institutional Next Generation Sequencing Core facility. The sequencing libraries were comprised of 50 bp long reads in a FASTQ format. The quality of each read was assessed and any base with a PHRED score of 15 or below was excluded from the analysis. In addition, the first 14 bases of each read were trimmed and any reads mapping to the human genome (version GRCh38/hg38) were excluded. The remaining 36 bp long reads were then mapped onto the R. prowazekii Breinl genome (NC_020993) allowing up to two base mismatches using Bowtie2  according to our standard protocol. The RNA samples were then treated with DNaseI (Zymo Research) and subjected to the MICROBEnrich Kit (Ambion). The RNA thus obtained were further purified via precipitation with 100% v/v ethanol prior to the samples determination of concentration in final preparations using a MultiSkan GO Microplate Spectrophotometer (ThermoScientific).HMECs infected with 32P] UTP-labeled RNA probes were synthesized through in vitro transcription with the MAXIscript® kit (Ambion)  following the manufacturer's instructions. Enriched bacterial RNA (15 μg per lane) was loaded onto a 1.5% agarose-formaldehyde gel, electrophoresed at 90 V, and then transferred onto a Zeta-Probe Blotting Membrane (Bio-Rad). Membranes were cross-linked using UV Stratalinker 1800 (Stratagene). A PCR template with a T7 promoter on the anti-sense strand for the sRNA under investigation was created using GoTaq® Green DNA Polymerase (Promega). Using the PCR template, strand-specific, 5, Rp_sR45, Rp_sR50, and Rp_sR66) were found to have sequence homology to other rickettsial species. All four had strong (~90%) homology to the typhus group R. typhi, while candidate Rp_sR5 also shared strong homology to R. felis, a transitional group species. Otherwise, all remaining trans-acting sRNAs were unique to R. prowazekii. With the exception of two candidates (Rp_sR3 and Rp_sR18), cis-acting sRNA candidates generally shared considerable homology to rickettsial species outside of the typhus group. The corresponding ORF for Rp_sR3 is H375_160 (a transcription-repair coupling factor), which is present in all other sequenced R. prowazekii strains. For Rp_sR18, the corresponding ORF is a single-stranded DNA-specific exonuclease (H375_870), which is also conserved in other R. prowazekii and R. typhi strains. Considering that the intergenic regions are more likely to undergo dynamic changes in their sequences and cis-acting sequences tend to remain conserved by virtue of their location on the anti-sense strand of an ORF, the high number of candidates sharing homology outside of the typhus group is not surprising. Accordingly, 17 cis-acting sRNAs were present on the ORFs conserved in all rickettsial species, and 4 sRNAs were encoded from an antisense strand representing hypothetical and ORFan proteins (ORFs with no known homologs within current databases). Interestingly, candidate Rp_sR12 had no homology to the spotted fever group, but it did share homology to the ancestral group, typhus group, and transitional group. Further, candidate Rp_sR28 had homology to the typhus group and transitional group, but no homology was found in the ancestral group or the spotted fever group. Candidate Rp_sR22 shared homology to the typhus group and the spotted fever group, but not the transitional group. It is important to note that homology was not necessarily observed for all rickettsial species of a particular group. For example, homology in candidate Rp_sR22 was found in the spotted fever group species R. montanesis and R. japonica, but not R. rickettsii or R. conorii.RNA-Seq based search of the transcriptome identified a total of 70 candidate trans-acting (intergenic) and cis-acting (antisense) sRNAs that were either expressed at 3 and/or 24 h post-infection Table . All seq32P] UTP-labeled RNA oligonucleotide probes specific to the corresponding candidate with enriched rickettsial RNA from R. prowazekii-infected HMECs at 3 and 24 h post-infection. Two trans-acting sRNAs (Rp_sR17 and Rp_sR60) and two cis-acting sRNAs (Rp_sR34 and Rp_sR47) identified from the RNA-Seq data were selected on the basis of their genomic location, number of RNA reads, strand orientation, as well as the orientation of corresponding upstream and downstream ORFs. Candidate Rp_sR67 [identified earlier with the SIPHT program as Candidate #25 , 4.5S RNA (ffs), RNaseP_bact_a, and α-tmRNA (ssrA). 6S RNA was only probed at 24 h post-infection as our previous data demonstrated that rickettsial 6S RNA was most abundantly expressed at this time-point  and 6S RNA, 5′ RLM-RACE was employed. RLM-RACE provides a distinct advantage over the traditional 5′ RACE method because it only amplifies primary transcripts, and not processed RNAs, for subsequent sequencing, which then permits the determination of an exact transcription start site. The TSSs for the novel R. prowazekii sRNAs, confirming the potential for their independent expression, are listed in Table Rp_sRs during infection of target host cells.Northern blot analysis only provides information regarding the levels of expression and the transcript size, but is unable to reveal the exact transcription start site. Therefore, to determine the transcription start sites of confirmed novel sRNAs , 150 nucleotides upstream of the identified transcription start site were submitted to the web-based program, BPROM. This range was chosen keeping in mind that ~80% of known σRp_sR17, Rp_sR60, and Rp_sR67). These programs identify the sRNA:mRNA interactions by assessing base pairing potential based on a Smith-Waterman dynamic and by assessing interaction sites and seed regions, respectively , transposon mutagenesis, and pseudogenization  is overrun by mobile genetic elements and conjugative plasmids resulting in acquisition of nearly 32% of the ORFan genes unique to REIS. It is presumed that large tracks of unique genes along with their IGRs may have been acquired from other unknown organisms with AT rich genomes similar to rickettsiae  at 3 and/or 24 h post-infection. In regards to the well-known sRNAs , there was clear expression at 3 and 24 h post-infection. The successful sequencing and determination of transcriptional start sites for each novel sRNA further demonstrates that R. prowazekii does, indeed, express these sRNAs during host cell infection. When the RLM-RACE data is overlaid with the RNA-Seq data, the transcription start sites, as determined by RLM-RACE, correspond to the start of the sequencing reads with the exception of Rp_sR60, in which case the transcription start site determined by RLM-RACE mapped toward the middle of the observed RNA-Seq reads. This could potentially be an outcome of: (i). occurrence of two novel small RNAs that may either have a significant overlap or may be located within extreme close proximity of each other in the R. prowazekii genome, (ii). presence of an unannotated ORF upstream of the sRNA TSS, and (iii). Rp_sR60 could be a riboswitch containing sRNA. Translation of the sequence upstream of the TSS identified by RACE revealed the existence of a predicted hypothetical ORF between positions 844285 and 844416. However, the –10 and –35 motifs were absent upstream of this predicted ORF. Riboswitch sRNAs are present in the 5′ untranslated region of the coding genes and regulate the expression of the downstream gene in response to metabolic stimuli. Several riboswitches have been well characterized in different bacterial species. Bartonella, a facultative intracellular pathogen, is shown to express at least 9 riboswitch sRNAs (Brt 1-9) upstream of different helix-turn-helix XRE genes   and spotted fever group  are not known to encode for hfq, nor has another chaperone molecule been described in Rickettsia species . Carboxyl-terminal proteases (CTPs) are a new group of serine proteases, including tail-specific proteases (TSPs), that have been recognized as critical players in bacterial protein processing . The T3SS is essential for cytotoxicity toward host cells and is a vital virulence factor in mouse models of acute pneumonia. Conversely, up-regulation of CtpA in P. aeuroginosa induces the expression of an extracytoplasmic function sigma factor regulon, resulting in an attenuated phenotype in rat models of chronic lung infection (Seo and Darwin, Brucella suis, ctpA-deficient strains display altered morphology, increased cell size, and partial dissociation of cell membrane from the envelope and are cleared 9 weeks post-inoculation in a BALB/c mouse model, suggesting that CtpA is critical for survival in macrophages (Bandara et al., Rp_sR47 and H375_3890, we hypothesize that Rp_sR47 interaction with H375_3890 may lead to the stabilization of the transcript for efficient translation. Further evaluation of this interaction and its functional implications in R. prowazekii are currently ongoing.Because cis-acting sRNAs exhibit perfect nucleotide similarity, the expected target would be the corresponding ORF on the opposite strand. In this context, H375_2470 as the corresponding ORF for R. prowazekii during in vitro infection of vascular endothelial cells, the primary target cell-type during human disease. We have further substantiated the potential implications of this new regulatory paradigm in rickettsial biology via determination of transcription start sites of select novel rickettsial sRNAs and predictive analysis of their target genes involved in the pathways of bacterial maintenance, growth, and survival, and mechanisms of pathogenesis. In summary, this study provides the very first glimpse of non-coding transcriptional landscape of R. prowazekii in target host cells.The field of bacterial pathogenesis is rapidly evolving and expanding with enhanced appreciation of the vastness of virulence factors and effector sRNAs. Here, we report on the identification of a number of sRNAs encoded and expressed by in vitro experiments and data analysis, and prepared the draft of the manuscript; HN conceptualized the idea of sRNAs in Rickettsia species and assisted with the performance and analysis of all aspects; MR, KK, and YF provided assistance with the details of bioinformatics and calculation of MEVs; AS and JP assisted with the preparation of rickettsial stocks and cultures of endothelial cells; RS assisted with in vitro experiments and data analysis; and SS conceived of the study and participated in its design and coordination and helped to finalize the manuscript. All authors read and approved the final manuscript.CS performed all bioinformatics analyses, This work was supported, in part, by a pilot project grant from the Institute for Human Infections and Immunity, the James McLaughlin Fellowship Program, and institutional support funds from the University of Texas Medical Branch, and an exploratory research grant AI115231-01A1 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health, Bethesda, MD, USA.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.


Adenosine-to-inosine (A-to-I) RNA editing, the most prevalent mode of transcript modification in higher eukaryotes, is catalysed by the adenosine deaminases acting on RNA (ADARs). A-to-I editing imposes an additional layer of gene regulation as it dictates various aspects of RNA metabolism, including RNA folding, processing, localization and degradation. Furthermore, editing events in exonic regions contribute to proteome diversity as translational machinery decodes inosine as guanosine. Although it has been demonstrated that dysregulated A-to-I editing contributes to various diseases, the precise regulatory mechanisms governing this critical cellular process have yet to be fully elucidated. However, integration of previous studies revealed that regulation of A-to-I editing is multifaceted, weaving an intricate network of auto- and transregulations, including the involvement of virus-originated factors like adenovirus-associated RNA. Taken together, it is apparent that tipping of any regulatory components will have profound effects on A-to-I editing, which in turn contributes to both normal and aberrant physiological conditions. A complete understanding of this intricate regulatory network may ultimately be translated into new therapeutic strategies against diseases driven by perturbed RNA editing events. Herein, we review the current state of knowledge on the regulatory mechanisms governing A-to-I editing and propose the role of other co-factors that may be involved in this complex regulatory process. This review discusses the current state of knowledge on the mechanisms adopted in regulation of Adenosine-to-Inosine RNA editing. A-to-I editing is a pivotal cellular process as demonstrated by mouse models, where ADAR1 knockout is embryonically lethal  and knocnrb . In addinrb .Mammalian ADAR family comprises three highly conserved members namely ADAR1, ADAR2 and ADAR3. ADAR members possess a conserved C-terminal deaminase domain and variable numbers of dsRNA-binding domains (dsRBDs)  Figure . Exclusi2CR) transcripts in vitro [in vivo evidence to prove beyond reasonable doubt that ADAR3 does not have any capacity to edit hitherto unknown ADAR substrates. With the advancements in RNA-sequencing technology, in vivo studies aiming to investigate the editing role of ADAR3 on an unbiased and global scale may come to fruition in the near future.The current consensus is that only ADAR1 and ADAR2 are catalytically active ,21 and tin vitro . RemarkaIntriguingly, previous observations noted that ADAR expression levels do not always correlate well with editing frequencies ,24. TherADAR1 exists in two predominant isoforms, p110 and p150, generated through transcription from alternative promoters . As depiIn the cell, trans-acting regulators determine the sub-cellular localization of ADAR1 and the p110 isoform localizes predominantly in the nucleus ,28. TranADAR1 expression is regulated on a post-transcriptional level by miRNAs, a class of small non-coding RNAs that regulate gene expression on the post-transcriptional level. In general, miRNAs, together with other players in the RNAi pathway, negatively regulate gene expression either by repressing translation or by promoting transcript degradation. miRNAs are approximately 22 nts in length and are at least partially complementary to the target sequences. In metastatic melanoma, it has been found that ADAR1 expression is frequently down-regulated by miR-17 and miR-432 .Intriguingly, ADAR1 exerts both pro-viral and anti-viral functions. Through the course of evolution, some viruses have evolved and acquired advantageous mechanisms favouring their own replication. For instance, adenovirus encodes two non-coding, virus-associated (VA) RNAs, known as VAI and VAII. The major form, VAI RNA, antagonizes deaminase activity of ADARs and may alter viral and cellular gene expression, mainly through modulation of RNA editing .418 attenuates ADAR1 editing activity without affecting its proper sub-cellular localization. It is hypothesized that SUMOylation of Lys418, a residue between the zDNA-binding domain and the first dsRBD of ADAR1, attenuates its editing activity through stereochemically hindering the binding of dsRNA and homodimerization of ADAR1, which are prerequisites for editing activities.ADAR1 has also been shown to be post-translationally modified through SUMOylation. This is a highly dynamic and reversible post-translational modification that involves covalent addition of small ubiquitin-like modifier (SUMO) on to the target protein. SUMOylation is involved in a myriad of cellular processes, including apoptosis, cell cycle regulation, protein stability and transcriptional regulation . Human AADAR2 gene expression can be stimulated by cAMP responsive element-binding protein 1 (CREB1) . In addiADAR2 negatively auto-regulates its activity through self-editing of its transcripts . ADAR2 c−/− mouse, ADAR2 was mislocalized in the cytoplasm of embryonic fibroblasts, which resulted in reduced editing at the GluR2 glutamine/arginine and arginine/glycine sites [ADAR2 sub-cellular localization is determined by trans-acting regulators. ADAR2 has a prominent nuclear localization characteristic as it lacks NES and contains a non-canonical NLS within the first 64 amino acid residues . Phosphone sites . This evne sites .Similar to ADAR1, ADAR2 shuttles dynamically between the nucleus and nucleolus and the presence of editable substrates initiates its shuttling between the two compartments . In the N-methyl-D-aspartate (NMDA) receptor which then initiates a cascade of events to ultimately activate the effector, calpain protease. The cleaved ADAR2 is now rendered non-functional due to its inability to bind to dsRNA substrates. The physiological impact of ADAR2 cleavage is pronounced. The decrease in GluRA2 glutamine/arginine editing causes a high calcium influx and excitotoxic neuronal death. However, it remains to be determined whether proteolytic cleavage of ADAR2 is a common mode of regulation in other non-neuronal cell types.Excitotoxic level of glutamate induces specific ADAR2 proteolytic cleavage between the two dsRBDs . This prOther factors that regulate ADAR2 have also been described. The crystal structure of ADAR2 unveiled a unique feature of its catalytic domain. A molecule of inositol hexakisphosphate (IP6) was found to be buried within the enzyme core of ADAR2 and is essential for proper protein folding and enzymatic activity of ADAR2 .Besides affecting nuclear localization of ADAR2, Pin1 also enhances the stability of ADAR2 proteins. ADAR2 mutants that can no longer interact with Pin1 were found to be less stable and the presence of Pin1 delays ADAR2 degradation . In contA heterologous editing assay that screens for editing regulators in yeast allowed the identification of ribosomal protein S14 (RPS14), Serine/Arginine-rich splicing factor 9 (SFRS9) and DEAH box polypeptide 15 (DDX15). Although the detailed regulatory mechanisms adopted by these proteins have not been studied, it has been proposed that the landscape of ribonucleoproteins (RNPs) affects RNA editing .Homodimerization of ADARs is necessary for RNA editing activity ,52. Howein vitro [in vitro experiments demonstrated that ADAR3 effectively suppresses editing of 5HT2CR RNA by ADAR1 and ADAR2 [ADAR3 remains to be a peculiar member of ADARs as it has no demonstrable editing activity in vitro . As mentnd ADAR2 . HoweverCapitalizing on the probable regulatory role of ADAR3 in A-to-I editing, it may be useful to understand the sub-cellular localization of this protein. Sub-cellular localization of ADAR3 is mostly nuclear as importin α1 specifically recognizes the R-motif to mediate nuclear transport . Till noRNA editing is a process under dynamic regulation. High throughput transcriptome sequencing revealed partially distinct A-to-I editing landscapes in brains of humans, chimpanzees and macaques . DespiteSpatiotemporal regulation of A-to-I editing further complicates the regulatory network in cells –61. SpecGiven the prominent effects of A-to-I editing on transcriptome, it is not surprising that aberrant A-to-I editing contributes to various diseases. Pioneering studies mainly focused on neurological functions, development and diseases, as A-to-I editing was first found to be a widespread process in the mammalian brain. These individual studies revealed that transcripts of numerous neurotransmitter receptors are precisely edited and dysregulated editing results in neurological diseases such as amyotrophic lateral sclerosis (ALS), transient forebrain ischaemia, Prader–Willi syndrome and psychiatric disorders . BesidesRecently, ADARs have emerged as promising therapeutic targets in cancer, a disease that is thought to arise from accumulation of various driver mutations that favour cell survival and proliferation. In the past, attention was on genomic mutations. However, increasing evidence demonstrates inconvertibly that mutations on the transcriptome level, owing to dysregulated RNA editing activities, have similar pathological effects. To date, perturbed A-to-I editing has been shown to be involved in various cancers, including acute leukaemia, breast cancer, hepatocellular carcinoma and neuroblastoma .The tight link between dysregulated A-to-I editing and diseases highlights the essentiality for a balanced regulation and regulators responsible for maintaining the balance are promising therapeutic targets.in vitro [The precise underlying regulatory mechanism of A-to-I editing has only been partially elucidated. In the present review, we reinforced the notion that ADARs, like many other crucial cellular components, are tightly regulated on transcriptional, post-transcriptional and post-translational levels . Althougin vitro ,65, it iin vivo RNA structures in a high throughput manner [Thus far, little advances have been made to investigate the substrate structures as RNA structome, the study of RNA structures, is technically challenging. With the introduction of selective 2′-hydrox acylation analysed by primer extension (SHAPE), coupled with RNA-sequencing technique, it might now be feasible to probe for t manner –68.In sum, our knowledge of A-to-I RNA editing is steadily increasing. Comprehensive delineation of the regulatory mechanisms underlying this pivotal process might eventually prove to be beneficial, especially in translational research. Promisingly, the invention of methods to correct specific RNA editing events –71 has o


PB2∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein  which activates type I interferon (IFN) responses through direct interaction with the adapter protein MAVS, a key component of the RIG-I-dependent IFN induction. This is the first time that such a function was described for a defective RNA-encoded protein, a finding that has several important implications with regard to deciphering viral protein functions and options for immunostimulatory approaches. Furthermore, this is an example of how influenza viruses may acquire novel polypeptides with altered functions from its limited genome.Error-prone polymerase function of RNA viruses can result in expression of defective RNAs harboring internal deletions of various sizes. Small subgenomic RNAs are strong inducers of the antiviral response by serving as pathogen-associated patterns that are predominantly detected by cellular sensors. Recently, it has been shown that influenza A virus defective RNAs are not only generated upon passages in cell culture, but also in infected humans, indicating that these subgenomic RNAs may also be relevant in infections  Orthomyxoviridae and are characterized by a segmented single-stranded RNA genome with negative orientation. These eight segments encode nine structural and up to seven nonstructural proteins -ATP (PerkinElmer) and T4 Polynucleotide Kinase (Fermentas) according to the manufacturer’s instructions. Northern blot analysis was performed as described previously [Total RNA (10 μg) was separated on 8% (w/v) polyacrylamide [29:1 acrylamide/bisacrylamide], 7 M urea gels and electro-transferred onto positively charged nylon membranes (Roche). Hybridization probes .All sequences were obtained from NCBI’s Influenza Virus Resource ((PDF)Click here for additional data file.S2 Tablehttp://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).All sequences were obtained from NCBI’s Influenza Virus Resource ((PDF)Click here for additional data file.S3 TableΔ-free rKAN-1 WT or Vietnam clones for 16–24 h. Undiluted supernatants were transferred to untreated MDCKII cells for 5 passages. Total RNA was isolated from every passage and expressional changes of PB2Δ RNA were analyzed by qRT-PCR and are depicted as n-fold normalized to first passage.MDCKII cells were infected with plaque-purified PB2(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 FigΔ RNA species were detected by qRT-PCR and are depicted as mean n-fold (±SD) of three independent experiments normalized to the respective RNA expression 2 h p.i..A549 cells were infected with 5 MOI KAN-1 and total RNA was isolated at the time points indicated. Expressional changes of the different PB2(PDF)Click here for additional data file.S2 Fig100 μg of protein mixtures from KAN-1-infected A549 cells were separated on a one-dimensional gel followed by in-gel digest by trypsin. LC-MS/MS analyses were performed on an EasyLC nano-HPLC (Proxeon Biosystems) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). The MS data were processed using default parameters of the MaxQuant software (v1.2.2.9).(PDF)Click here for additional data file.S3 FigΔ for 2, 4, 6 or 8 h. Expression of viral proteins PB1, PB2, NP, M1 and NS1 was analyzed by Western blot. Presence of PB2Δ was verified by using PB2-specific antibodies. ERK2 expression served as loading control. Blots are representative of three independent experiments.A549 cells were infected with 5 MOI of rKAN-1 WT or PB2(PDF)Click here for additional data file.S4 FigΔ-expressing virus. A) Expression changes of IFNβ mRNA in lungs were analyzed two days p.i. by qRT-PCR. N-fold expression in individual animals normalized to uninfected PBS control mice is depicted. B) Viral lung titers 2 days p.i. of individual animals are depicted. A, B) Statistical significance was analyzed by Mann-Whitney test.BALB/c mice were infected with 50 pfu rKAN-1 wild type or PB2(PDF)Click here for additional data file.


Paramphistomum leydeni and Paramphistomum cervi are the same or distant species.Rumen flukes parasitize the rumen and reticulum of ruminants, causing paramphistomiasis. Over the years, there has been considerable debate as to whether P. leydeni was amplified using PCR-based sequencing and compared with that of P. cervi. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) of P. leydeni specimens (n = 6) and P. cervi specimens (n = 8) was amplified and then sequenced. Phylogenetic relationship of the concatenated amino acid sequence data for 12 protein-coding genes of the two rumen flukes and selected members of Trematoda was evaluated using Bayesian inference (BI).In the present study, the complete mitochondrial (mt) genome of P. leydeni was 14,050 bp in size. Significant nucleotide difference between the P. leydeni mt genome and that of P. cervi (14.7%) was observed. For genetic divergence in ITS-2, sequence difference between P. leydeni and P. cervi was 3.1%, while no sequence variation was detected within each of them. Phylogenetic analysis indicated that P. leydeni and P. cervi are closely-related but distinct rumen flukes.The complete mt genome of P. leydeni and P. cervi represent two distinct valid species. The mt genome sequences of P. leydeni provide plentiful resources of mitochondrial markers, which can be combined with nuclear markers, for further comparative studies of the biology of P. leydeni and its congeners from China and other countries.Results of the present study support the proposal that The online version of this article (doi:10.1186/s13071-015-0823-4) contains supplementary material, which is available to authorized users. Paramphistomum (Trematoda: Digenea), known as the ‘rumen flukes’ or ‘amphistomes’, are the pathogens of paramphistomiasis of ruminants, such as cattle, buffalo, sheep, goat and deer ; Fasciola hepatica (NC_002546) , tRNA genes (13.20%) and non-coding regions [LNCR (38.33%) and SNCR (35.94%)] . The difference between the two Paramphistomum spp. is similar to that between F. hepatica and F. gigantica [D. chinensis and D. dentriticum [C. sinensis and O. felineus [P. leydeni and P. cervi are different Paramphistomum species.Phylogenetic analysis of the concatenated amino acid sequence datasets for all 12 mt proteins Figure  reflecteigantica , D. chintriticum , and C. felineus  by obserP. leydeni samples was approximately 2582 bp in length, including partial 18S rDNA, complete ITS-1, complete 5.8 rDNA, complete ITS-2, and partial 28S rDNA. ITS-2 was 286 bp in length. Sequence difference in ITS-2 rDNA was 3.1% between the P. leydeni and P. cervi, which is slightly lower than that between D. chinensis and D. dentriticum (3.8-6.3%), but higher than that between F. hepatica and F. gigantica (1.7%) [P. leydeni and P. cervi. These results provided additional strong support that P. leydeni and P. cervi are different trematode taxa.The rDNA region sequenced from individual a (1.7%) , while nParamphistomum species, elaborate population genetic investigations still need to be conducted. Further studies could (i) explore nucleotide variation in mtDNAs among Paramphistomum populations in various hosts of numerous countries from different continents, (ii) establish accurate molecular tools and rapid detection methods, (iii) decipher the genomes of Paramphistomum using next generation sequencing (NGS) technologies. It is believed that elucidating the transcriptomes, proteomes and genomes of Paramphistomum would assist in future efforts in deciphering biology and taxonomy of more trematode parasites including the important family Paramphistomatidae.In spite of the evidence of genetic difference between two P. leydeni, and provided reliable genetic evidence that P. leydeni and P. cervi are closely-related but distinct paramphistome species based on mt and nuclear ribosomal DNA dataset. The accurate identification of the two rumen flukes will contribute to the diagnosis and control of paramphistomiasis. The availability of the complete mt genome sequences and nuclear rDNA sequences of P. leydeni could provide additional genetic markers for studies of the epidemiology, population genetics and phylogenetic systematics of trematodes.The present study determined the complete mt genome sequences and ITS-2 rDNA sequences of