Trypanosoma cruzi specific in vitro transcription (IVT) linear mRNA amplification method in order to improve parasite transcriptomics analyses.Trypanosomatids are a group of protozoan parasites that includes the etiologic agents of important human illnesses as Chagas disease, sleeping sickness and leishmaniasis. These parasites have a significant distinction from other eukaryotes concerning mRNA structure, since all mature mRNAs have an identical species-specific sequence of 39 nucleotides at the 5′ extremity, named spliced leader (SL). Considering this peculiar aspect of trypanosomatid mRNA, the aim of the present work was to develop a T. cruzi SL sequence, bearing an upstream T7 promoter (T7SL primer), which was used to direct the synthesis of second-strand cDNA. Original mRNA was then amplified by IVT using T7 RNA polymerase. T7SL-amplified RNA from two distinct T. cruzi stages (epimastigotes and trypomastigotes) were deep sequenced in SOLiD platform. Usual poly(A) + RNA and and T7-oligo(dT) amplified RNA (Eberwine method) were also sequenced. RNA-Seq reads were aligned to our new and improved T. cruzi Dm28c genome assembly (PacBio technology) and resulting transcriptome pattern from these three RNA preparation methods were compared, mainly concerning the conservation of mRNA transcritional levels and DEGs detection between epimastigotes and trypomastigotes.We designed an oligonucleotide complementary to the last 21 bases of T7SL IVT method detected more potential differentially expressed genes in comparison to either poly(A) + RNA or T7dT IVT, and was also able to produce reliable quantifications of the parasite transcriptome down to 3 ng of total RNA. Furthermore, amplification of parasite mRNA in HeLa/epimastigote RNA mixtures showed that T7SL IVT generates transcriptome quantification with similar detection of differentially expressed genes when parasite RNA mass was only 0.1% of the total mixture (R = 0.78 when compared to poly(A) + RNA).The T7SL IVT amplification method presented here allows the detection of more potential parasite differentially expressed genes (in comparison to poly(A) + RNA) in host-parasite mixtures or samples with low amount of RNA. This method is especially useful for trypanosomatid transcriptomics because it produces less bias than PCR-based mRNA amplification. Additionally, by simply changing the complementary region of the T7SL primer, the present method can be applied to any trypanosomatid species.The online version of this article (10.1186/s12864-017-4163-y) contains supplementary material, which is available to authorized users. Transcriptomics is a relatively new area of life sciences that aims to analyze the complete set of expressed RNAs in a specific cell or organism, under specific conditions, using high-throughput techniques . InitialThree main RNA amplification methods are mostly used: (i) in vitro transcription (IVT), (ii) PCR-based and (iii) rolling circle amplification (reviewed by ). IVT prTrypanosoma cruzi), sleeping sickness (Trypanosoma brucei) and leishmaniasis (Leishmania sp) [T. brucei life cycle [T. brucei-specific cDNA on host-parasite RNA mixtures, followed by PCR amplification using nested primers to amplify parasite mRNA. Although introducing significant bias in comparison to poly(A) + RNA, the authors considered that PCR based amplification method of SL-containing mRNAs allowed comparison of different samples as long as they were all treated equally [Trypanosomatids are a group of human pathogenic parasites that includes the etiological agents of Chagas disease (ania sp) . Trypanoania sp) . Throughania sp) . This chania sp)  enrichesania sp) –27 and tfe cycle , 26. Detfe cycle , 29 and fe cycle . Recentlfe cycle  used SL  equally .T. cruzi SL sequence for parasite-specific mRNA amplification. We demonstrate a T. cruzi specific IVT amplification method based in second-strand cDNA synthesis using a SL primer bearing an upstream T7 promoter (T7SL). RNA-Seq analyses of two distinct forms of T. cruzi (epimastigotes and trypomastigotes) show that T7SL amplification does not introduce significant bias in RNA-Seq quantification and also allows parasite transcriptome analysis in mixed RNA samples. This method presents better performance, measured by Pearson correlation, than the recently published PCR-based T. brucei mRNA amplification [Here, we describe the use of fication , enablinT. cruzi Dm28c epimastigotes [2 atmosphere using a multiplicity of infection (MOI) of 10 parasites per host cell. Trypomastigotes were recovered after 4 days of infection, in the cell burst peak.Parasite culture: stigotes  were culstigotes . Tissue T. cruzi cells [5) epimastigotes were sorted directly to 700 μl of RNA extraction buffer (buffer RLT of Qiagen RNeasy kit) kept under 4 °C during sorting. Total sorting volume was ~200 μl. To the resulting cell lysate (900 μl), 250 μl of 100% ethanol was added and RNA was purified by RNA clean-up protocol of Qiagen handbook with additional on-column DNase digestion step.Flow cytometry and cell sorting experiments were performed in a FACSAriaII  using a 85 μm nozzle and 45 psi sheet pressure. A total of 10,000 events were acquired in the regions previously identified as corresponding to zi cells . For epiT. cruzi epimastigote and trypomastigote forms (5 × 108 cells) with the RNeasy kit , according to the manufacturer’s instructions, with an additional on-column DNase digestion step. Total RNA from HeLa cells were obtained from MessageAmp™ II aRNA Amplification Kit from Life Technologies® (#AMB1751-5). RNA integrity was assessed on an Agilent 2100 Bioanalyzer with RNA 6000 Nano LabChip kit, according to the manufacturer’s instructions.Total RNA was extracted from ® mRNA Isolation System III from Promega® (#Z5300), according to the manufacturer’s instructions.Polyadenylated RNA were purified from at least 50 μg of total RNA using PolyA + Track® (#AMB1751-5). For classic Eberwine amplification method, first-strand cDNA was synthesized using a T7oligo(dT) primer that contains a T7 RNA polymerase promoter upstream to the poly-T tract; this promoter will further direct the in vitro mRNA synthesis (amplification). First, 100 ng of total RNA (in 11 μl) were mixed with first-strand cDNA reaction mix containing 1 μl of T7oligo(dT) primer, 2 μl first-strand buffer, 4 μl dNTPs, 1 μl RNase inhibitor and 1 μl ArrayScript Reverse Transcriptase and incubated for 2 h at 42 °C. Second-strand cDNA was then synthesized for 2 h at 16 °C using the total 20 μl first-strand product plus 80 μl of second strand mix containing 63 μl water, 10 μl second-strand buffer, 4 μl dNTPs, 2 μl DNA polymerase and 1 μl of RNase H. Double-strand cDNA was then purified using PureLink® PCR Micro Kit from Life Technologies (#K310250) according to manufacturer’s instructions. Eluted cDNA was adjusted to 16 μl and used as template for amplification reaction in a total volume of 40 μl containing 16 μl NTPs, 4 μl amplification buffer and 4 μl of T7 RNA polymerase; in vitro transcription took 14 h at 37 °C. Amplified RNA (aRNA) was then purified using RNeasy MinElute CleanUp Kit , according to manufacturer’s instructions.All mRNA amplification reactions were done using reagents from MessageAmp™ II aRNA Amplification Kit from Life TechnologiesT. cruzi spliced leader (in bold) with an upstream T7 RNA polymerase promoter (in lower case) (5′-ggccagtgaattgtaatacgactcactatagggaGGCGGTACAGTTTCTGTACTATATTG-3′), which we named T7SL. In this case, T7SL was used for second-strand cDNA synthesis, while first-strand cDNA was produced using random primers, according to the second round amplification protocol of MessageAmp™ II aRNA Kit. Template total RNA was adjusted to 10 μl, mixed with 2 μl of “second round primers” (random primers) and incubated at 70 °C for 10 min followed by snap cooling on ice to allow annealing of random primers to the RNA. Then, 8 μl of the reverse transcription master mix was added and first-strand cDNA was synthesized at 42 °C for 2 h. RNase H was added (1 μl) and the sample incubated at 37 °C for 30 min to specifically degrade the remaining RNA. T7SL primer was added to a final concentration of 1 μM and the sample was incubated at 70 °C for 10 min and then placed on ice. Second-strand cDNA master mix without RNase H was added (74 μl) and the reaction incubated for 2 h at 16 °C. Double-strand cDNA purification, in vitro transcription and aRNA purification was performed as described above.For specific amplification of spliced leader containing mRNA, we used a custom designed primer complementary to the last 21 bases of ® Pfx DNA Polymerase  for second-strand cDNA synthesis at 68 °C instead of 16 °C, and primer annealing at a higher temperature, when using the DNA polymerase of MessageAmp™ II aRNA Kit. In this case, after RNA degradation with RNase H, single-strand cDNA were mixed with T7SL primer (1 μM) and Platinum® Pfx DNA polymerase mix  in a final volume of 50 μl. Sample was placed on a thermal cycler as following: 95 °C for 3 min (denaturing), 55 °C for 10 min  and 68 °C for 30 min (second-strand cDNA synthesis). cDNA purification, in vitro transcription and aRNA purification was performed as described above.For a more stringent second-strand cDNA synthesis directed by T7SL primer, we also developed a modified protocol using Platinum® 2.0 fluorometer (Life Technologies). For length distribution analysis, aRNA concentration was adjusted to 1 ng μl−1 and analyzed on an Agilent 2100 Bioanalyzer with RNA 6000 Pico LabChip kit, according to the manufacturer’s instructions.Amplified RNA was quantified using Qubit RNA HS Assay Kit (#Q32852) in a QubitNegative control aRNA samples, generated from pure HeLa RNA, were sequenced in Omega Bio-Tek Inc.  using the Illumina 2 × 100 paired-end sequencing method. All other samples were sequenced in the DNA Sequencing platform of Instituto Carlos Chagas  using SOLiD™ 1 × 50 single-end sequencing method.® PCR Micro Kit. Amplified DNA was quantified with Qubit® dsDNA HS Assay Kit on a Qubit® 2.0 fluorometer. Equal masses of each sample, containing specific barcodes, were pooled together and the mixture used as DNA template for emulsion PCR on Applied Biosystems SOLiD® EZ Bead™ E80 system (#4453095). After 3′ end modification with terminal transferase, template beads were deposited onto glass slides and libraries sequenced on a SOLiD™ 4 system using multiplex fragment sequencing protocol that generates about 700 millions 50 bases short reads per run.For SOLiD™ sequencing, Total RNA-Seq Kit  was used accordingly to manufacturer’s instructions. Briefly, RNA was fragmented using RNase III and cleaned up with Invitrogen RiboMinus™ Concentration Module. Strand-specific adaptors were hybridized and ligated to both extremities of RNA in an overnight reaction, followed by first-strand cDNA synthesis with reverse transcriptase. cDNA was purified using MinElute® PCR Purification Kit (Qiagen) and gel-based selected for size using a Novex TBE-Urea 6% gel (#EC6865bOX). On-gel cDNA was amplified by PCR using SOLiD™ RNA barcoding kit primers  and purified with Invitrogen PureLinkT. cruzi Dm28c genome (GenBank accession number MBSY00000000) and human genome (version 19) using Bowtie2 v. 2.3.0 [Illumina 2 × 100 paired-end RNA-seq reads were aligned to the v. 2.3.0  in localT. cruzi genome is highly repetitive [SOLiD 1 × 50 single-end reads were aligned using SHRiMP2 v. 2.2.3  with thepetitive , 38; mulT. cruzi gene annotation and read counts, clusters of orthologous genes were determined by MCL algorithm v. 10-148, with an inflation value –I of 5.0 [2 of SG read counts.For RNA-Seq gene coverage analysis, genes were divided in 100 equally proportional bins and the number of reads aligned to each bin were summed up from the SAM file  using cuI of 5.0 , based oI of 5.0  and diffI of 5.0 , using aI of 5.0 . Pearson−5. The present results and Li and collaborators [To compare our transcriptome data to that from Li and collaborators , we tookborators  data werAll RNA-Seq data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE94766.T. cruzi spliced leader with an upstream T7 RNA polymerase promoter (see methods), which we named T7SL. First-strand cDNA was synthesized using random primers and, after RNA degradation, second-strand cDNA was produced using the T7SL primer  profile with the aRNA generated by the classic Eberwine method that uses T7oligo(dT) primer. For specific amplification of mRNA bearing SL, we designed an oligonucleotide complementary to the last 21 bases at the 3′-end of mer Fig. . Distincmer Fig. . Hence, T. cruzi, both amplification methods showed a smaller median RNA size  + RNA. We sequenced an average of 24 ± 4 million short reads 50 bases) for each one of the 44 samples in the SOLiD™ platform  [RNA-seq reads were aligned to our first version of (PacBio) . As this(PacBio) . These iT. cruzi genome, per Supra Gene read counts were computed. When comparing the technical replicates , both IVT amplification methods have a very high correlation between technical replicates , T7SL showed a higher number of DEGs, as this method detected 56.3% and 6.5% more putative DEGs in comparison to poly(A) + and T7dT methods  IVT and poly(A) + RNA) to compare two different stages of ods Fig. . However2) for all parasite genes, and showed that epimastigote to trypomastigote fold changes are similar for all three methods  , but in order to increase specificity, we modified the protocol used for T. cruzi RNA samples, using Platinum® Pfx DNA Polymerase for second-strand cDNA synthesis at 68 °C instead of 16 °C when using regular DNA polymerase. This modification did not affect the yield and pattern of T. cruzi RNA amplification  900 ng HeLa plus 100 ng Epi (10% parasite RNA), (ii) 990 ng HeLa plus 10 ng Epi (1% parasite RNA) and (iii) two mixtures containing 0.1% of parasite RNA (4995 ng HeLa plus 5 ng Epi and 9990 ng HeLa plus 10 ng Epi). The 0.1% mixtures corresponds to about 106 human cells  together with 104 epimastigotes .We conducted RNA-Seq experiments on aRNA generated from mixture samples and the resulting reads were aligned to a ome Fig. ; for ther = 0.77). This result indicates that T7SL amplification method performs better, in general, than PCR based mRNA amplification, since a recent study in T. brucei based on this technique reported a correlation of 0.38 [When comparing read counts per Supra Gene for pure and mixed samples using T7SL Pfx IVT aRNA Fig. , the par of 0.38  between T. cruzi-HeLa mixtures containing only 0.1% of epimastigote RNA, T7SL amplification generate a similar coverage throughout the genome , the great majority of them (~80%) are mapped in or nearby a 186 nt repetitive element that are close to the rDNA gene cluster  to Poly(A) + RNA. In general, the global RNA quantification distribution is maintained, hence even with few nanograms of RNA  we can quantify the parasite transcriptome with satisfactory precision . Results showed that even when pushing down the input mass to 3 ng, the transcriptome quantification shows a very good correlation  to the trypomastigote stage, as a reference sample. Besides, we included the limiting mass samples on the same analysis. After detecting DEGs in all comparisons to trypomastigote, we plot in a heat map the 1079 most confident ones accordingly the optimal T7SL condition: epimastigote  versus trypomastigote (T7SL)(FDR 0.01 and twofold change). Interestingly, Fig. 5 parasites. Comparisons (ii) to (v) were against T7SL IVT trypomastigote aRNA. After independent calculation of DEGs for each comparison, we compare the list of DEGs (considering an FDR of 1%) and plotted the overlap of DEGs in an Euler diagram  purified poly(A) + RNA obtained for both parasite stages (gold standard RNA-Seq method), (ii) pure epimastigote aRNA produced from 100 ng of total RNA, (iii) the most limiting mass used for T7SL IVT (3 ng of epimastigote RNA), (iv) the lower percentage of epimastigote RNA in a host-parasite mixture (0.1% of parasite RNA) and (v) epimastigote RNA obtained by sorting of 10ram Fig. . A vast T. cruzi [T. brucei [T. cruzi transcriptomic modulation associated with the establishment of intracellular T. cruzi infection. In the initial times of infection (4 to 24 h), when the number of parasites to host cells is not higher than 1:1, generally less than 5% of the mixed host-parasite read pool mapped to the T. cruzi genome, reflecting the larger proportion of the host cell transcriptome. Hence, in cases where the relative number of parasites to host cells is much smaller than 1:1, the fraction of reads mapping to parasite genome will be too small. In this scenario, the parasite specific mRNA amplification method showed here is of great utility. The T. cruzi/HeLa mixed samples showed that it is possible to quantify the parasite transcriptome even when only 0.1% of total RNA mass correspond to parasite RNA, which is similar to a mixture of 106 human cells and 104 T. cruzi epimastigotes. In this case, typical poly(A) + RNA-Seq will not efficiently capture the parasite transcriptome and the present method is especially useful for low parasitemia samples or low input parasite total RNA. Considering that all mRNAs from trypanosomatids possess the same specific sequence at the 5′ region, the spliced leader or mini-exon, they could be selectively amplified through a SL-bearing primer. Although the SL trapping method theoretically provides a means of analyzing parasite transcriptome in complex RNA samples (such as host-parasite mixtures) [T. brucei-specific cDNA by priming with a SL-specific oligo, followed by PCR amplification using nested primers to evaluate the T. brucei transcriptome in host-parasite RNA mixtures consisting of only 0.1% of T. brucei RNA. Their PCR based amplification of SL-containing mRNAs allowed comparison of different samples as long as they were all treated in the same way, but introduced significant bias when comparing to gold standard poly(A) + RNA-Seq [Direct RNA-Seq analysis of parasites in their hosts is a difficult task due to the low amount of RNA available to be extracted directly from infected tissue in comparison to the higher amount of co-purified host RNA. When the parasitemia is relatively high, typical poly(A) + RNA-Seq can be used to simultaneously capture the parasite and host cell transcriptomes, as recently showed for T. cruzi , 48, T. . brucei  and Leis. brucei . Li and . brucei  analyzedion 4 to  h, when ixtures) . Using a RNA-Seq .r = 0.77, for the 0.1% mixture) in comparison to PCR-based method (r = 0.34) [Our method uses a different procedure, IVT for RNA amplification, as it linearly amplifies original mRNAs . As alre = 0.34) . This imThe higher ability to detect epimastigote versus trypomastigote DEGs when using samples produced by the T7SL IVT method corroborate the idea that linear RNA amplification enables a more reproducible transcriptome quantification , 13, 17.Other useful characteristic of the T7SL IVT method is the 5′ bias of the generated reads. Due to the fact that 5′-UTR regions are significantly smaller than 3′-UTR , usuallyWe concluded that the T7SL IVT method has several advantages for those researchers preparing trypanosomatid RNA-Seq libraries whenever the parasite RNA mass is a concern. As long as all samples are treated equally, T7SL IVT allows a powerful detection of putative DEGs in complex samples, including low input and host-parasite mixtures .T. cruzi total RNA as template for amplification, this method can be easily adapted for any trypanosomatid species by only modifying the T7SL oligonucleotide. T7SL IVT opens new perspectives for trypanosomatid studies, specially parasite transcriptome analysis directly in their hosts.Although we developed T7SL IVT method using Additional file 1:T. cruzi cluster of orthologous genes, named as Supra Genes (SG) in this article. The CDS nomenclature (TcCLB) is from T. cruzi CL Brener genome annotation. (XLSX 278 kb)Shows the description of each Additional file 2:Shows the correspondence of each Dm28c Supra Gene to CL Brener Esmeraldo haplotype ID here used to compare the present work with that of Li and collaborators . Additional file 4:Contains all supplemental figures  and tables (Table S1 and S2). (DOCX 1811 kb)Additional file 5:Contains raw and normalized SG reads counts for all RNA-Seq samples. Detailed descriptions of all sample names are shown in Additional file Additional file 6:Contains lists of epimastigote to trypomastigote DEGs for each method used in the present article . (XLSX 1545 kb)


Transposable elements, once described by Barbara McClintock as controlling genetic units, not only occupy the largest part of our genome but are also a prominent moving force of genomic plasticity and innovation. They usually replicate and reintegrate into genomes silently, sometimes causing malfunctions or misregulations, but occasionally millions of years later, a few may evolve into new functional units. Retrotransposons make their way into the genome following reverse transcription of RNA molecules and chromosomal insertion. In therian mammals, long interspersed elements 1 (LINE1s) self-propagate but also coretropose many RNAs, including mRNAs and small RNAs that usually exhibit an oligo(A) tail. The revitalization of specific LINE1 elements in the mammalian lineage about 150 Ma parallels the rise of many other nonautonomous mobilized genomic elements. We previously identified and described hundreds of tRNA-derived retropseudogenes missing characteristic oligo(A) tails consequently termed tailless retropseudogenes. Additional analyses now revealed hundreds of thousands of tailless retropseudogenes derived from nearly all types of RNAs. We extracted 2,402 perfect tailless sequences (with discernible flanking target site duplications) originating from tRNAs, spliceosomal RNAs, 5S rRNAs, 7SK RNAs, mRNAs, and others. Interestingly, all are truncated at one or more defined positions that coincide with internal single-stranded regions. 5S ribosomal and U2 spliceosomal RNAs were analyzed in the context of mammalian phylogeny to discern the origin of the therian LINE1 retropositional system that evolved in our 150-Myr-old ancestor. Long interspersed element 1 (LINE1 or simply L1) autonomous retrotransposons evolved less than 450 Ma in the common ancestor of deuterostomes, a clade comprising Echinodermata and Chordata, and, over time, gave raise to more than 300 diverse element families and a plethora of subfamilies . Frequencis-retrotransposition of their own RNA, but also retrotransposed any RNAs equipped with at least an oligo(A) tail. This included many SINEs, such as Alu elements  of nonautonomous elements  promoter sequences and an ∼300-nt-long polyadenylated 3′-UTR. The two open reading frames (ORFs) encode a functionally largely, as yet, uncharacterized but essential RNA binding protein with nucleic acid chaperon activity (ORF1)  and a prelements . Intereselements .The molecular mechanism of LINE retrotransposition, known as target primed reverse transcription (TPRT), mediates the reverse flow of genetic material from cellular RNA back to the genome . Active A and B). Besides all mechanistic requirements, element-specific activity patterns and population structures, two major factors influence the occurrence and frequency of inheritable genomic tailless retropseudogenes: 1) The germline expression rate of source RNAs and 2) the activity of the relaxed L1 element that occasionally compete for limited host factors with other autonomous retrotransposons. At least two probably active retropositional systems are known in cow, elephant, and marsupials . In lizards, many different systems compete for retrotranspositional dominance . In the human genome, L1s are the only active autonomous LINE retrotransposons, and nearly 100 of them are still capable of retrotransposition . Activity assays have demonstrated that six source loci produce more than 80% of all retrocopies and are classified as hot L1s (The oligo(A) tail length of pol III transcripts appears to be crucial for successful retroposition events . In contfig. 2 hot L1s . Tailles hot L1s . With thhttp://www.repeatmasker.org/RMDownload.html, last accessed March 11, 2015). Human sources included predicted tRNAs from the Genomic tRNA Database , LINEs , histones, monoexonic and housekeeping mRNAs , 18S rRNA (K03432), 28S rRNA (M11167), 5.8S rRNA (U13369), 5S rRNA (X51545), Y RNAs , 7SK RNA (NR_001445.2), U1, U4, U5, U6, U7, and U13 , U2 (K03022), U4atac (NR_023343.1), U6atac (NR_023344.1), U11 (NR_004407.1), U12 (NR_029422.1), snoRNAs U3, U8, U14, U17 , and others from snoRNAbase , the sources of 5S rRNA and U2 are listed in the supplementary tables S9 and S10, Supplementary Material online. In cases where consensus sequences were not available, we generated a Consensus60 sequence after BLASTN searches for full-length elements with the 5S rRNA or U2 snRNA sequence of the closest relative species or the vertebrate consensus sequence from the RepeatMasker library  as input.To detect different types of tailless retropseudogenes genome wide, we chiefly screened the human genome for all known small RNAs, ribosomal RNA genes, and some mRNA subsets, including LINEs, histone, monoexonic mRNA, and housekeeping genes, compiled in a user-defined library using the local version of RepeatMasker , we filtered the RepeatMasker results for 5′- complete  and 3′-truncated (at least 10 nt) hits flanked by 8–35 nt TSDs (with at least 70% sequence identity) and a target sequence identity to the consensus sequences of more than 69%. To further compensate for random mutations, we allowed a 15 nt gap between the 3′-element terminus and the TSD start. The minimal length of a tailless retropseudogene was set to 30 nt. For searches of tRNA-derived tailless retropseudogenes, we used more relaxed conditions that permit 20 nt between the 5′-terminal TSD and the element start in order to account for potential precursor sequences.With a designed Python script  was used to identify TSDs according the following settings: Gap penalty −25, match +8, mismatch −17, and applying the substitution matrix S11 . In comparison, the global Needleman Wunsch algorithm was employed for alignment of the tailless RNA hits with the corresponding source RNA. Here, the following settings were elaborated: Match +7, mismatch −5, gap opening penalty −25, gap extension penalty −3, and applying the substitution matrix S12 . From the ∼2,500 human hits with TSDs, we eliminated all multiple hits of the same genomic region  and genomic duplications. A genomic duplication was identified in cases where TSDs and the ∼100-nt flanking sequences were 70% or more identical. The Python script relied on the same Smith Waterman settings introduced above. Interrupted elements wrongly assigned as tailless elements were detected and excluded by screening the RepeatMasker output files for identical repeat-specific IDs. Finally, we received 2,402 clear instances of tailless retropseudogenes from different reverse-transcribed RNAs. The same procedure was applied to screen all other vertebrate genomes and plants in this study. To ensure data comparability, the number of tailless retropseudogenes per genome was normalized per gigabase genome sequence.The “tailless” Python script used different kinds of alignment generations. The local Smith Waterman algorithm  to search for 5S rRNA and U2 snRNA hits (full-length and tailless) flanked by simple repeats or low complexity regions at respective 3′-ends.To uncover possible RNA pol III-transcribed (tailless) retropseudogenes with additional, so far unknown, polyadenylation, we used the human RepeatMasker output , using only elements with 100% identical TSDs. To enable unbiased comparison of both 3′-full-length and 3′-tailless elements , we utilized identical numbers of either case. Thus, we screened RepeatMasker output files for 1,183 randomly selected LINE/L1 and SINE/Alu repeats (human), and LINE/L1 and B1 elements (mouse). The designed Python script (TSDfinder.py) permits an overlap with the elements 3′-end of maximally 20 nt as a priming site. The length of TSDs ranged between 8 and 35 nt. There was no discrimination of a minimal or maximal insert length, but the 3′-ends were required to be not truncated . To calculate the target site pattern, 2 nt upstream of the 5′-TSD and the 5′-TSD itself were considered. After counting the number of nucleotides for each position, the connected frequency site distribution was calculated to build a sequence logo motive with the help of the R seqLogo package , extracted the corresponding priming sites (if available), and calculated their respective length. The common target site pattern of LINE and SINE elements, so far described as TT/AAAA , was rec package . The calTo calculate the “breakpoint” patterns, we used all human tailless elements with 100% identical TSDs and their corresponding RNA targets. We selected regions 5 nt upstream and downstream relative to the breakpoint within the target RNA and counted the frequency of each nucleotide per position. The depiction as sequence logo was conducted as described above.http://www.girinst.org/ last accessed March 11, 2015), and then screened the outfile for 4′-full-length  and 5′-truncated (at least 5 nt of nonmatching sequences), 3′-tailless (at least 10 nt of nonmatching sequences) and 3′-full-length  elements with the TSDfinder Python script introduced above. Furthermore, we demanded sequence identities of at least 70% for the respective TSDs.To compare the numbers of tailless and full-length L1-dependent integrations, we masked the human genome for L1 elements  corresponding to the methods described in http://www.repeatmasker.org/genomicDatasets/RMGenomicDatasets.html, last accessed March 11, 2015; RepeatMasker Library 4.0.5 20140131) of human (hg19), mouse (mm10), dog (canFam3), cow (bosTau7), sloth (choHof1), elephant (loxAfr3), tenrec (echTel2), manatee (triMan1), rock hyrax (proCap1), opossum (monDom5), wallaby (macEug2), Tasmanian devil (sarHar1), platypus (ornAna1), chicken , and lizard (anoCar2).For http://www.bioinformatics.uni-muenster.de/tools/gpac, last accessed March 11, 2015; http://www.repeatmasker.org/genomicDatasets/RMGenomicDatasets.html, last accessed March 11, 2015).We used the online genome-wide “Presence/Absence Compiler”  (B), which differs slightly from the canonical TT/AAAA (A). The latter sequence was reconfirmed by random extraction and analysis of equal amounts of L1 and Alu elements with perfect TSDs and full-length 3′-ends . Interestingly, the consensus sequence for tailless retropseudogene insertions carries an A or T (W) nucleotide at the break position and continues with four A’s and two additional W’s. X(0–17) represents a more heterogeneous complementarity of the insertion site internally base paired with the corresponding template RNA. The variant motifs suggest that the processes of retroposition of 3′-oligoadenylated RNAs and tailless retropseudogenes vary somewhat between classical and tailless retroposition.Tailless retropseudogenes also use TPRT for retroposition, but presumably are primed internally at loops or other single-stranded regions of source RNAs ((0–17) B, which  TT/AAAA  L1 conse TT/AAAA A. The laC). On the basis of the assumption that truncated RNAs resulting from endonucleolytic processing or degradation represent bona fide targets for RNA polyadenylation and subsequent retroposition, we scanned the entire human genome for 5S rRNA and U2-derived retropseudogenes. The corresponding retropseudogenes should then harbor oligo(A) tails at sites of theoretical truncations. However, we failed to identify any convincing signals consistent with truncated RNAs serving as intermediates for tailless retropseudogene generation. Furthermore, we unsuccessfully scanned human small RNA transcriptome libraries for 5S rRNA and U2 RNA-derived cDNAs, whose 3′-termini would be consistent with the truncation of source RNAs as templates for tailless major breakpoint (data not shown). By default, this strongly suggests internal priming as the actual scenario.In general, there are two entirely different scenarios that might lead to the generation of tailless retropseudogenes with preferential 3′-endpoints C. On thesupplementary table S1, Supplementary Material online).Internal priming of dimeric Alu SINEs could well explain the “revived” activity of long-extinct Alu monomeric elements . Insteadsupplementary table S2, Supplementary Material online). Furthermore, we detected 51 5′-truncated tailless histone retropseudogenes derived from mRNAs, known for their terminally nonpolyadenylated mRNAs .There is an additional trace in our data set indicating that a terminal oligo(A) tail is not essential for retrotransposition. For example, RNA pol III-transcribed 7SK RNA, which participates in the control of RNA pol II transcription during elongation, is not polyadenylated but was the source of 139 tailless retropseudogenes within our data set , we found no tailless element related to the highly abundant nucleolar RNAs.Usually, the interaction of RT/EN protein and L1 mRNA constituting the RNP complex competent for retrotransposition takes place within the cytoplasm. This is also the bona fide site for the recruitment of SINE source RNAs related to tRNA, 5S rRNA, and 7SL RNA, such as, for example, B2, ID, B1, and Alu. Such SINE transcripts are significant sources for tailless retropseudogenes as well (data not shown). It is expected that binding of source RNA during LINE RNP formation might preferentially occur within the cytoplasm. However, our data revealed that nuclear RNAs, such as U6 RNA , unprocehttp://www.bioinformatics.uni-muenster.de/tools/gpac, last accessed March 11, 2015) derived presence/absence patterns for our 2,454 human tailless retropseudogenes . Selection of GPAC patterns with clear presence/absence boundaries in all investigated primates yielded 19 human-, 26 Homininae-, 36 Hominidae-, 38 Catarrhini-, 258 Anthropoidea-, 15 Haplorrhini-, and 14 primate-specific insertions . supplementary table S5, Supplementary Material online), display a similar distribution pattern as tailless elements.Three major factors determine the level of genomic fixation of tailless retropseudogenes in germ lines: 1) The expression rate of the source RNAs and their capacity to offer templates to retroposition over time, 2) the L1 activity over time, and finally 3) population structures and speciation events. Given the unique genomic information available for human and at least substantial genomic information for most other primate lineages, the ∼63 Myr of primate evolution  are wellsupplementary file S1 and tables S2 and S6, Supplementary Material online), perfectly agree with the activity range of L1 in some vertebrates and reach highest abundance in Boreotheria . Currently, we have no explanation for the marked difference between 5S rRNA tailless retropseudogenes in mouse  versus human . The higher number of 5S rRNA tailless retropseudogenes in the guinea pig  resembles more closely that of human. Despite a low abundance of tailless retrotransposons, the elephant has an enormous number of lineage-specific full-length L1 elements . In contrast, the relatively closely related manatee has only six full-length versions of L1 elements. In afrotherians, RTEs represent the dominant retrotranspositional system, as indicated by coretroposed AfroSINEs , indicating possible additional restrictions of random RNA retroposition.Transcription and reverse transcription are the two major processes that any successful autonomous or nonautonomous master element has to undergo to be propagated. During the course of evolution, the activities of individual LINE and SINE subfamilies rise and fall in successive waves, due to, for example, changing measures of host defense and/or alteration of the retropositional efficiency in case of individual master genes. The presence of SINEs, processed retropseudogenes, and tailless retropseudogenes is a strong indication for successful L1 activity. With inactivation of the vigorous LINEs, the nonautonomous elements depending on them concomitantly are silenced. One prominent example of this dependency is a mammalian-wide interspersed repeat that coevolved with LINE3 elements and then ceased to retropose in mammalian genomes about 130 Ma concurrent with the “perishing” of the partner LINE3 system . L1 actifroSINEs . In marsfroSINEs . There ifroSINEs , we deteFrom a rough selection of about 450,000 human 3′-truncated tailless retropseudogene hits, we selected 2,402 clear cases with flanking TSDs (without genomic duplications) derived from various template RNAs . This se4 or more copies, today we know that retroposition is an ancient process, and that the mostly neutrally evolving products, including SINEs, LINEs, and mRNA-derived retrogenes, decay by mutational attrition over a time span of a few hundred million years. A few members of very ancient SINE families, however, persisted. For example, from the Amniota SINE1 family, about 1,000 copies are still discernible in the human genome, and no one questions, despite their relatively low copy number, that they are SINEs.Singer first introduced the abbreviations SINEs and LINEs for short and long interspersed sequences, respectively, in 1982 , and alsOrnithorhynchus anatinus. Usually, snoRNAs are transcribed by RNA pol II, often located in introns of protein-coding host genes and processed to yield the mature product.Reverse transcription is primed mainly by a recessed DNA end (3′ overhang) on RNA as a template, but it does not seem to matter whether this priming occurs on the oligoadenosine-rich 3′-end of a transcribed master SINE (preferred) or internally (on occasion), which leads to 3′-truncated “SINEs” that still feature TSDs. Copy numbers in the range of about 600 are reported for this latter group . Clearlyhttp://www.girinst.org, last accessed March 11, 2015). Like so often in biology, borders are hard to delineate on a continuum, in this case also underscored by the blurred distinction of RNA pol II and III promoter elements  are L1 mobilized, 3′-truncated RNA descendants with an overlapping phylogenetic distribution to their L1 drivers. There are not only tRNA-derived elements, but also high copy derivatives of 5S rRNA, U2 RNA, 7SK RNA, and many more. Here, we describe 2,402 of the most unambiguous cases of tailless retroposons detected in the human genome. The abundance of different types of tailless retropseudogenes somewhat corresponds to the expected expression activity of their source RNAs. In primates, they accumulated most successfully in the common ancestor of anthropoid primates . A similar distribution occurred for independently derived ERV–LTR insertions, indicating that population bottlenecks and/or a long anthropoid ancestry are more probable causes of this lopsided accumulation than variation in L1 activity. Because our stringent search criteria were limited to perfect cases, we present only a small fraction of the actual number of tailless retropseudogenes. The large number of tailless retrotransposons confirms the previously debated process of generating 3′-truncated tailless retropseudogenes, and provides convincing arguments against the previously proposed strict necessity of oligo(A) tails for L1-mediated retrotransposition and instead exemplifies an extensive, somewhat adenosine-rich complementarity during internal priming. The process of generating tailless retropseudogenes dates back to the common Jurassic ancestor of therian mammals and to the evolution of a L1 retroposition machinery that is more relaxed with respect to template RNAs.Supplementary figure S1, tables S1–S12, and files S1 and S2 are available at Genome Biology and Evolution online .


Vaccination, namely the controlled activation of antigen-specific immune cells, is a first and critical step toward engendering protective immunity against cancer. A large number of vaccination strategies developed to date, from simple to complex, have shown but limited efficacy. In a recent article in Nature, Kranz and colleagues describe what appears to be a remarkably effective and relatively simple approach, using tumor antigen encoding mRNA complexed to cationic lipid carriers, RNA-lipoplexes (RNA-LPXs) like DOTMA, DOPE and DOTAP . Vaccina+ DC, induces IFNα, and stimulates a T cell response [The main finding was that the intravenously injected negatively charged anionic, but not the conventional neutral or positively charged cationic mRNA lipoplexes were taken up almost exclusively by CD11c + macrophages and dendritic cells (DC) residing in the spleen, lymph nodes and other organs like the liver. This was accompanied by exceptionally strong and durable T cell responses unlike what has been previously seen. The mechanism was dependent on macropinocytosis-mediated uptake of the RNA-encoded antigens by the DC. Macropinocytosis, which is constitutively active in immature DC and downregulated upon their maturation, is unique in its ability to efficiently promote the translocation of its cargo to the cytoplasm, and plays a key role in cross-presentation of exogenous class II, and probably class I, antigens. Uptate of the RNA-LPXs by the DC led to a TLR7 dependent induction of IFNα presumably triggered by the encapsulated mRNA, which led to their maturation and ability to optimally activate T cells. Local and transient expression of IFNα is a key mediator promoting the induction of potent T cell immunity against blood-borne pathogens  and tumoresponse .This rather simple and cost-effective vaccination protocol using readily available reagents offers a number of important advantages: (i) It dispenses with the need to develop and use a targeting ligands to suitable receptors like DEC205 or Clec9A that are expressed on the surface of resident DC . (iv). EUltimately the question is whether the RNA-LPX vaccine will elicit clinical responses in cancer patients that are superior to what we have seen so far. In transplantable murine tumor models vaccination in prophylactic and therapeutic settings suggests that the approach has merit, but the conditions were not particularly stringent so the potency was difficult to asses. More impressive were early results from an ongoing clinical trial in melanoma patients. Three patients treated with low to moderate doses of the RNA-LPX vaccine targeting 4 melanoma antigens exhibited robust and durable T cell responses, in one patients reaching levels comparable to that of CMV and EBV T cell responses that are thought to be protective. Such responses appear to be more robust than what has been generally seen in similar settings but it is hard to judge. Evaluation of more patients, treated with higher doses of vaccine, and signs of clinical responses, are eagerly awaited.It is prudent to assume that the RNA-LPX vaccination as a stand alone therapy will not be the sought for “magic bullet”, but rather an important component in a multi-pronged approach. One challenge is to improve the vaccine induced immune response, expanding the vaccine activated T cells thru 4-1BB, OX40, GITR or CD27 costimulation, and extending its persistence by skewing the differentiation of the vaccine-activated T cells to become long lasting memory cells . Not lesInitial evidence from the murine studies and the ongoing clinical trial is that the RNA-LPX vaccine will be safe. There may be some risk that the systemically administered RNA-LPX could elicit excessively high levels of IFNα by the CD11c + cells scattered throughout the body that could provoke autoimmune sequalae especially in predisposed individuals. This can be minimized by targeting the RNA lipoplexes to the tumor lesions using suitable ligands decorating the lipoplexes that bind to tumor secreted products such as VEGF or osteopontin . Tumor t


The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min.Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 × 10 Coronaviridae, subfamily Coronavirinae) can infect a wide variety of animals, including humans, avians, rodents, carnivores, chiropters and other mammals, and can cause respiratory, enteric, hepatic, and neurological diseases , real-time reverse transcription PCR (rRT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-LAMP . HoweverFigure 1).In the RT-LAMP-VF assay, the RNA of MERS-CoV was amplified by RT-LAMP using multiple cross-linked primers (six primers) which target the conserved region of N gene. Two loop primers (LF and LB) which are involved in isothermal amplification are labeled with fluorescein isothiocyanate (FITC) and biotin, respectively. After amplification, the amplicons are simultaneously labeled with FITC and biotin. The amplicons that are labeled with biotin can bind to colloidal gold particles conjugated with streptavidin to form a complex. Then the complex, labeled with FITC, is captured by an anti-FITC antibody that is coated on the text line of the strip, and the test results are presented as a colored line that is visible to the naked eye  of MERS-CoV was kindly provided by professor Jincun Zhao from the Guangzhou Institute of Respiratory Health . The ChiThe intestinal tissues of bats infected with SARS-related (SARSr)-CoV or HKU4 were gifted by professor Changchun Tu from the Institute of Military Veterinary Medicine. RNA was extracted using a commercial RNA extraction kit  in the BSL2 Laboratory.Figure 2. A 213-nt region among the complete RNA positions 28848–29061 of the EMC strain (GenBank Accession No. JX869059) was selected for submission to an online primer design software (Primer Explorer V4).1 Six specific primers were designed in eight regions of the gene (Table 1) and synthesized by Shanghai Invitrogen Biological Engineering, Co., Ltd.In total, the N gene sequences from 35 representative complete MERS-CoV strains from previous studies that were collected from different regions between 2012 and 2015 were aligned. Due to the picture size limit, alignment results that included 19 MERS-CoV strains, 2 SARS-CoV strains, 1 HKU4 strain, 1 HKU5 strain, and 2 SARSr-CoV strains were presented in 23× 10-9× concentration/(fragment length × 660). We obtained the recombinant plasmid concentration of 370 ng/μl, which corresponded to 7.9 × 1010 copies/μl, and 10-fold serial dilutions of the recombinant plasmid ranging from 107 to 100 copies/μl were prepared.A pcDNA3.1 (+) recombinant plasmid (build by predecessors of our team) containing the MERS-CoV N gene (GenBank Accession No. JX869059) was measured with a spectrophotometer, and the copy number of the recombinant plasmid was calculated using the following formula: copies/μl = 6.02 × 10in vitro transcription by Bao Biological, Co., Ltd., Dalian, China. The RNA transcripts were then purified and quantified, and the copy number was calculated using the following formula: copies/μl = 6.02 × 1023× 10-9× concentration/(fragment length × 340). The concentration of the synthesized RNA transcript was 1760 ng/μl as measured with a spectrophotometer, which corresponded to 9.7 × 1012 copies/μl. The synthesized RNA transcripts were stored at -80°C after purification.The MERS-CoV N gene target sequence (GenBank Accession No. JX869059) was synthesized using 4, 0.2 μM of FIP and BIP, 0.1 μM of LF and LB, 0.05 μM of F3 and B3, 1.4 μM of deoxynucleotide triphosphates (dNTPs) (Thermo Scientific), 0.2 M of betaine and 8 U of Bst DNA polymerase (New England BioLabs) were used. If the recombinant plasmid was used as the template, the mixture  was heated to 95°C for 5 min and then immediately placed on ice. Next, 8 U of Bst DNA polymerase was added, and the mixture was incubated at 65°C for 50 min. If the synthesized RNA transcripts were used as the template, 5 U of avian myeloblastosis virus reverse transcriptase (AMV)  was additionally added to the reaction mixture described above, and the mixture was incubated at 65°C for 50 min.The reactions were prepared as previously described . BrieflyThe reactions were performed at five different temperatures  for 50 min, and the results were analyzed with the vertical flow visualization strip  to obtain the optimal reaction temperature. Three replications were performed for each trial.Based on the optimum reaction temperature, the reaction time  and inner primer concentration (0.2 or 0.4 μM) were simultaneously optimized. Three replications were performed for each trial.Because this method was ultimately applied to detect MERS-CoV RNA, the recombinant plasmid was replaced with synthesized RNA transcripts as the amplification template. The reaction process was executed in a thermostatic water bath at varying temperatures  for 30 min. Three replications were performed for each trial.TMsp RP Multimarker Controls kit . The pathogens that made up RP Multimarker 1 (RP1) and RP Multimarker 2 (RP2) controls included HKU-1, OC43, NL63, 229E, influenza A/B, rhinovirus, adenovirus, and parainfluenza, etc. (Table 2). RNA of the RP1 controls, RP2 controls, SARSr-CoV and HKU4 and synthesized RNA transcripts were used to evaluate the specificity of the RT-LAMP-VF assay. Three replications were performed for each trial.Multiple respiratory pathogen nucleic acids were extracted from the NATtrol6 to 2 × 100 copies/μl) were subjected to the RT-LAMP-VF assay to assess the detection limits. Three replications were performed for each trial.Ten-fold serial dilutions of the synthesized RNA transcripts . The primes and probe were designed and synthesized by TaKaRa Biotechnology, Co., Ltd., China  approved all the experimental procedures.Table 3 revealed that the highest amplification efficiency occurred at 65°C. Analysis of the results revealed no significant differences in three replications. Therefore, 65°C was deemed the optimum temperature for the RT-LAMP-VF assay.First, recombinant plasmids were used as the template to optimize the RT-LAMP-VF assay reaction conditions. To screen for the optimum temperature, a denaturation step was initially performed at 95°C for 5 min, and the reaction was then incubated at five different temperatures  for 50 min. Table 4, the same results were obtained at incubation periods of 30 and 40 min in equivalent conditions. Thus, 30 min was selected as the optimal reaction time. Simultaneously, the primer concentrations were optimized as presented in Table 4, and 0.2 μM for FIP/BIP, 0.1 μM for LF/LB and 0.05 μM for F3/B3 were deemed optimal.To determine the optimal amplification time for the RT-LAMP-VF assay, recombinant plasmids were detected at three different reaction times , and the application signal was monitored by the strip. As presented in Table 5, the best sensitivity and specificity were obtained when the reaction was performed at 61°C.The feasibility of the RT-LAMP-VF assay was further confirmed using synthesized RNA transcripts in the optimized reaction conditions. As presented in Figure 3, only the synthesized RNA transcripts tested positive, and multiple respiratory pathogen nucleic acids from RP1 and RP2, SARSr-CoV, and HKU4 all tested negative with the RT-LAMP-VF assay. Therefore, the RT-LAMP-VF assay had no cross-reactivity with multiple CoVs, including SARSr-CoV, HKU4, HKU1, OC43 and 229E, etc.Living organisms infected by respiratory pathogens often exhibit atypical respiratory symptoms. To accurately screen MERS-CoV positive patients with respiratory symptoms, multiple respiratory pathogen nucleic acids produced from RP1 and RP2, SARSr-CoV RNA, HKU4 RNA and synthesized RNA transcripts were used to evaluate the specificity of the RT-LAMP-VF assay. As illustrated in 6 to 2 × 100 copies/μl) were used to assess the sensitivity of the RT-LAMP-VF assay. The assay limit of detection for synthesized RNA transcripts was 2 × 101 copies/μl within 35 min. The representative results of the RT-LAMP-VF assay for serial dilutions of synthesized RNA transcripts were presented in Figure 4.Next, 10-fold dilutions of synthesized RNA transcripts  were detected with the RT-LAMP-VF assay. Negative results were observed when the concentration was lower than 1 × 101 copies/μl .Ten-fold serial dilutions of MERS-CoV RNA (ranging from 1 × 105 to 1 × 10-1 copies/μl) were spiked into the total RNA of the throat swabs collected from healthy people, and the mixtures were used to further evaluate the RT-LAMP-VF assay. This assay did not react non-specifically with nucleic acids from the throat swabs. Additionally, the limit of detection was equivalent to 1 × 101 copies/μl of MERS-CoV RNA , which indicated that the total throat swab RNA did not affect the specificity or sensitivity of the RT-LAMP-VF assay for detecting MERS-CoV.Different copy numbers of viral RNA (ranging from 1 × 103–1 × 10-1 copies/μl) were used as the templates in conventional RT-LAMP, rRT-PCR and RT-LAMP-VF assays. The positive reaction was clearly observed for the 1 × 102 copies/μl of MERS-CoV RNA sample using 2% agarose gel electrophoresis in the conventional RT-LAMP reaction . The limit of detection was equivalent to 1 × 101 copies/μl of MERS-CoV RNA in the RT-LAMP-VF . Moreover, both of the rRT-PCR assays could detect templates as little as 1 × 100 copies/μl of MERS-CoV RNA for the upE or N2 gene , and these results are consistent with those of previous reports.The diluted MERS-CoV RNAs  are labeled with FITC or biotin, creating the visible results on the test strip.The optimal amplification temperatures were different for the recombinant plasmids and synthesized RNA transcripts, which could have been due to several reasons. First, the RT-LAMP-VF assay requires AMV Reverse Transcriptase to reverse transcribe the RNA into cDNA. AMV Reverse Transcriptase exhibits its highest activity at 42–55°C, while Bst DNA polymerase operates best at 65°C. The higher reaction temperature likely decreased the efficiency of reverse transcription. Therefore, when synthesized RNA transcripts were used as the template, the temperature was lowered to facilitate reverse transcription. Furthermore, the supercoiled structures of the recombinant plasmids were steady, thus making it necessary to maintain the helix state at a high temperature.According to a study by 1 copies/μl of MERS-CoV RNA for the RT-LAMP-VF, which is more sensitive than the conventional RT-LAMP reaction (1 × 102 copies/μl). Here, we observed high sensitivities, the two rRT-PCR assays detected as litter as 1 × 100 copies/μl in both the upE and N2 assays. Although the sensitivity is lower than that of the rRT-PCR, the RT-LAMP-VF assay only requires 35 min, which is far faster than the rRT-PCR assay, which requires 2 h. Moreover this method does not require electronic equipment to read the signal of the amplification products.The rRT-PCR assay is recommended by the WHO for the routine confirmation of cases of MERS-CoV infection. In this study, the two rRT-PCR assays were guided by the study reports of Because the collected clinical MERS samples were mostly sputum, we needed to verify that the RT-LAMP-VF assay did not react with sputum nucleic acids and that the sputum samples did not affect the assay sensitivity. However, clinical specimens infected with MERS-CoV are difficult to obtain in non-endemic countries such as China. The mixtures of the total RNA of throat swabs and different copy numbers of MERS-CoV RNA were subjected to the RT-LAMP-VF assay. Our results indicated that the sensitivity and specificity of this method were not interfered with by other components in the clinical sample.In summary, our study demonstrates that the RT-LAMP-VF assay enriches the practical tools that are available molecular diagnosis of MERS-CoV. The RT-LAMP-VF assay for MERS-CoV exhibited no cross-reactivity with multiple CoVs, which demonstrated good specificity. Additionally, this assay’s rapidity and lack of need for special instrumentation indicates that it can be easily adapted to different laboratory settings.HW, SY, and XX designed the experiments. PH, HJ, ZC, HC, FY, XH, FW, CJ, PFH, SX, YZ, JW, WS, and TW performed the experiments. HW, JZ, BY, NF, YG, SY, and XX analyzed the data. PH, HW, and HJ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TS and handling Editor declared their shared affiliation.


Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV. Western equine encephalitis virus (WEEV), Eastern equine encephalitis virus (EEEV), and Venezuelan equine encephalitis virus (VEEV) are arthropod-borne (arbo) viruses of the genus Alphavirus of the virus family Togaviridae. To date these viruses are restricted to the Americas but due to worldwide travelling and trade they might be introduced also to other parts of the world in the future. All three equine encephalitis viruses are classified as Category B agent by the Centers for Disease Control and Prevention, Atlanta (https://emergency.cdc.gov/agent/agentlist-category.asp). They are transmitted by sanguivorous mosquitoes within bird ) or rodent populations , respectively. Infections in reservoir hosts do not lead to obvious clinical signs. However, severe diseases can occur when equines and humans are infected with epizootic subtypes by biting mosquitoes. In the last decade 662 equine cases with 302 fatalities of VEE were reported to the OIE and 75,000–100,000 human cases with more than 300 fatalities were counted in the most recent outbreak in Venezuela and Colombia in 1995 [http://www.oie.int/). In the time period 1964 to 2009 the CDC registered 639 human WEEV and 260 EEEV cases with only few fatalities. It was shown recently in an experimental animal model that all three viruses are transmissible by aerosols [In the time period 2007 to 2012 a total of 1926 EEEV and 3 WEEV associated equine cases were diagnosed in the United States  for EEEV and WEEV were published –13. Howequencing . Recent quencing , 16. In Multiple sequence alignments of VEEV sequences were performed using Vector NTI Advanced v.10  and MEGA Software  to reveaRT-qPCR was carried out by using a commercial kit . After the reverse transcription (50°C for 30 minutes) the DNA was denatured (95°C for 15 min). Amplification cycles included denaturation (95°C for 15 sec), annealing (55°C for 30 sec), and elongation (72°C for 30 sec) steps. Ct values were determined by the CFX96 software . XbaI and subsequently transcribed into RNA and the DNA degraded using the Riboprobe® Combination System, T3/T7 RNA Polymerase , and the QIAamp Viral RNA Mini Kit (Qiagen) was used for RNA isolation (without carrier RNA). The RNA concentration was estimated with the Quant-It™ RNA Assay Kit, Broad-Range (Invitrogen). The copy number of the synthetic RNA was calculated from the RNA concentration and the molecular mass of the RNA transcript.To determine the copy number of viral genomes a synthetic calibrator was developed, which comprises a T7 RNA polymerase promoter and the target sequences for the RT-qPCRs of EEEV, WEEV, and VEEV  cloned i2 to 1010 copies. Based on the corresponding standard curves the sensitivity and viral load for different EEV strains in concurrent runs could be determined: the limit of detection (LOD) corresponded to 66,2 copies per μL  for VEEV (TC-83 strain), at 736 copies  for WEEV (McMillan strain), and at 316 copies per μL  for EEEV (New Jersey strain)  are considered to be positive. VEEV specific primer/probe combination did not detect other equine encephalomyelitis viruses   . Since sV, WEEV)  or close in vitro as aforementioned. All VEEV subtypes were successfully detected by the novel RT-qPCR assay with a suitable sensitivity and high performance as demonstrated by linear standard curves over 5 logs  encompassing the target region and representing 10 different VEEV subtypes . This inr 5 logs . R2 valficiency . To evalWe report here the first experimental evidence of a quantitative real-time RT-PCR assay for the sensitive and specific detection of all known VEEV subtypes or sequence variants. The synthetic calibrator RNA allows the determination of viral genome equivalents of VEEV as well as WEEV and EEEV by a one-step RT-qPCR reaction.


We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the immunological surveillance of cytotoxic CD8+ T cells.Despite eliciting a potent CD8 Brucella abortus is one of the intracellular bacterial species that cause brucellosis, a zoonotic worldwide disease. An intricate enigma of Brucella immunity is its long-term persistence inside host despite a vigorous and specific immune response. Our study describes a novel immune evasion strategy exploited by B. abortus: the down-modulation of the IFN-γ-induced expression of the molecules responsible for antigen presentation to CD8+ T cells on the surface of monocytes, Major Histocompatibility Complex Class I (MHC-I) molecules. We found that the bacterial component responsible for this phenomenon is its RNA, a component associated with bacterial viability itself. Specifically, we demonstrated that not only does intact RNA down-modulate MHC-I but also the RNA degradation products. Bacterial RNA is sensed by pattern recognition receptors (PRRs), among which TLRs family has gained more attention. In this study, we demonstrated that the receptor involved in this phenomenon is TLR8, a single-stranded RNA and RNA degradation products sensor. By means of this mechanism, Brucella impairs antigen presentation to CD8+ T cells, hiding within infected cells and avoiding the immunological surveillance of cytotoxic cells. On balance, these results provide new evidence to understand how B. abortus can survive inside the host and persist chronically. Brucella spp. [+ T cells are determinant to restrain Brucella infection. The importance of these cells resides in their capacity to eliminate Brucella-infected target cells [+ T cells are developed during Brucella infection [Brucella-infected macrophages to present bacterial antigens on MHC-I molecules and activate cytotoxic CD8+ T cell responses. Despite this immune response, Brucella is able to persist inside these cells establishing a chronic infection. Therefore, as a successful chronic and persistent pathogen, Brucella must own an effective strategy to subvert the challenge of highly outfitted CD8+ T cells. We have previously reported that infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression. As a consequence, B. abortus-infected macrophages display diminished capacity of antigen presentation to CD8+ T cells [B. abortus to induce the retention of these molecules within the Golgi apparatus [B. abortus involved in this phenomenon remained unknown.Host control of brucellosis requires a set of cells and components of the immune system which together promote a complex response against lla spp. . Yet, fret cells , 3. Prevnfection , 4, conf T cells . MHC-I dpparatus . HoweverB. abortus-mediated MHC-I down-modulation is dependent on bacterial viability as was demonstrated by the incapacity of heat-killed bacteria to inhibit the expression of such molecules [B. abortus mutant strains devoid of key virulence factors, B. abortus RB51 (a rough LPS mutant) and B. abortus virB10- (a VirB type IV secretion system mutant), are capable of inhibiting the IFN-γ-induced MHC-I surface expression to the same extent as wild-type B. abortus [B. abortus mutant strains are unable to persist inside human monocytes for a long period despite their preserved capacity of infecting cells [Interestingly, olecules . Further abortus . These Bng cells –9. Consing cells . Overallvita-PAMPs), among which prokaryotic RNA is included [It has been recently demonstrated that the immune system is capable of sensing the most essential characteristic of microbial infectivity, microbial viability itself . The immincluded , 11.Recognition of nucleic acids in general and RNA in particular by receptors of the innate immune system is a complicated and interesting field of investigation. The immune system must discriminate between ‘self’ (host) and ‘foreign’ (invading pathogen) nucleic acids . This prB. abortus involved in the inhibition of MHC-I could be vita-PAMPs such as B. abortus RNA, since they are found exclusively in live bacteria and are actively expressed during early stages of infection. Thus, the aim of this study was to characterize the components, signaling pathways and mechanisms implicated in MHC-I down-modulation. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages.Taking our previous results into account, we hypothesized that the components of B. abortus-mediated MHC-I inhibition is dependent on bacterial viability [B. abortus rough LPS mutant RB51 and a mutant in the B. abortus type IV secretion system VirB, two mutant strains in key virulence factors, are capable of inhibiting the IFN-γ-induced MHC-I surface expression to the same extent as wild-type B. abortus [B. abortus mutant strains on key virulence factors and evaluated whether their live and heat-killed (HK) forms could inhibit the IFN-γ-induced MHC-I surface expression on THP-1 cells. The mutant strains used were: RB51 (rough LPS mutant), virB10- (mutant in VirB type IV secretion system), btpA, btpB single mutants and a btpAbtpB double mutant , and Bpe159  and all mutant strains studied were able to diminish the IFN-γ-induced MHC-I surface expression in a dose-dependent manner after 48 h. However, this phenomenon occurred exclusively when bacteria were alive (9 bacteria/ml) ; its outer membrane protein 19 (Omp19), a prototypical lipoprotein of B. abortus, on its lipidated (L-Omp19) and unlipidated (U-Omp19) versions and B. abortus DNA (Ba DNA). None of the evaluated structural components was able to inhibit the IFN-γ-induced MHC-I surface expression , as it is present only in viable bacteria [B. abortus RNA was the component involved in the inhibition of MHC-I surface expression on human monocytes, we used B. abortus WT RNA to stimulate THP-1 cells in the presence of IFN-γ for 48 h at different concentrations. The expression of MHC-I was then evaluated by flow cytometry. B. abortus WT RNA significantly down-regulated the IFN-γ-induced surface expression of MHC-I molecules in a dose-dependent manner . This treatment was not able to down-modulate MHC-I . Furthermore, B. abortus RNA treatment did not induce early and late apoptosis or necrosis as determined by the Annexin V assay, even at the highest evaluated concentration (10 μg/ml)  . On the B. abortus (HKBA) to inhibit MHC-I surface expression, we determined by gel electrophoresis that HKBA lacked RNA and that the products recovered from the HKBA RNA extraction process were unable to inhibit MHC-I surface expression . Confirming these results, the endocytosis inhibitor Nystatin was able to reverse MHC-I inhibition mediated by stimulation with B. abortus RNA  were able to inhibit MHC-I surface expression. On the contrary, eukaryotic RNA  was unable to inhibit MHC-I surface expression, even at the highest concentration utilized . After that, we verified that the treatments with the enzymes had not affected the integrity of the RNA  were stimulated with different concentrations of t manner . Thus, BBrucella RNA to form secondary structures is still unknown and considering that TLR3 has been implicated in many functions mediated by viral double-stranded RNAs, we first wanted to evaluate whether TLR3 could be involved in B. abortus RNA-mediated inhibition of MHC-I molecules. TLR3 is the unique TLR that transduces its signal through the adapter protein TRIF. We therefore evaluated the effect of B. abortus RNA in BMM from TRIF KO mice. B. abortus RNA was able to inhibit the IFN-γ-induced MHC-I surface expression in BMM from TRIF KO mice to the same extent as in BMM from WT mice (B. abortus RNA we used a TLR3 inhibitor (TLR3/dsRNA Complex Inhibitor). Yet in the presence of a TLR3 inhibitor, B. abortus RNA down-regulated MHC-I expression confirming that TLR3 is not involved in this phenomenon . Neutralization of the EGFR significantly reversed the inhibition of MHC-I surface expression mediated by ORN06  for 48 h, followed by incubation with OVA peptide (SIINFEKL) and a B3Z T-cell hybridoma specific for OVA-Kb, which carries a β-galactosidase construct driven by NF-AT elements from the IL-2 promoter. BMM from WT and TLR7 KO mice treated solely with mIFN-γ presented Kb-restricted OVA peptide efficiently after 6 h and onwards (B. abortus RNA (1–10 μg/ml) in the presence of mIFN-γ significantly inhibited presentation of OVA peptide since it diminished the response of B3Z cells, compared to mIFN-γ-only treated cells  were generated by differentiation of bone marrow progenitors from female C57BL/6 mice (aged 2–3 months). Mice were kept under specific pathogen-free conditions in positive-pressure cabinets and provided with sterile food and water ad libitum. All animal procedures were performed according to the rules and standards for the use of laboratory animals of the National Institutes of Health. Animal experiments were approved by the Animal Care and Use Committee of the IMEX Institute. The protocol license number assigned by this Committee is: 020/2016.B. abortus S2308, Salmonella typhimurium SL14028, Bacillus cereus B10502, Escherichia coli 11105 and Klebsiella pneumoniae 700603, and B. abortus RB51, B. abortus virB10, B. abortus btpA, B. abortus btpB, B. abortus btpAbtpB or B. abortus Bpe159 mutant strains were cultured in tryptose-soy agar supplemented with yeast extract (Merck). The number of bacteria on stationary-phase cultures was determined by comparing the OD at 600 nm with a standard curve. To obtain heat-killed B. abortus strains, bacteria were washed in PBS, heat killed at 70°C for 20 min and stored at -70°C until used. Total absence of B. abortus viability subsequent to heat killing was verified by the absence of bacterial growth in tryptose-soy agar. All live Brucella manipulations were performed in biosafety level 3 facilities, located at the Instituto de Investigaciones Biomédicas en Retrovirus y SIDA .Lipoproteins were expressed and purified as previously described . To elimB. abortus 2308 LPS was provided by I. Moriyón . The purity and characteristics of these preparations have been described elsewhere [B. abortus DNA was purified by extraction with phenol:chloroform:isoamyl alcohol and ethanol precipitation [lsewhere . LPS waspitation . To elim2 atmosphere and standard medium composed of RPMI-1640 supplemented with 25 mM Hepes, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Gibco), 100 U of penicillin/ml and 100 μg of streptomycin/ml. THP-1 cells were obtained from the American Type Culture Collection  and cultured as previously described [3 (EMD Millipore) for 72 h. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque  gradient centrifugation from human blood collected from healthy adult individuals. Monocytes were then purified from PBMCs by Percoll  gradient and resuspended in standard medium. Purity of the isolated CD14+ monocytes was more than 80% as determined by flow cytometry. Viability of cells was more than 95% in all the experiments as measured by trypan blue exclusion test. Mouse bone marrow-derived macrophages (BMM) were generated by differentiation of bone marrow progenitors from C57BL/6 wild type mice, TRIF KO or TLR7 KO mice  with rM-CSF (PeproTech), as previously described [All experiments were performed at 37°C in 5% COescribed . To induescribed .6/ml were infected in round-bottom polypropylene tubes  with different multiplicities of infection (MOI) of B. abortus S2308, B. abortus RB51, B. abortus virB10, B. abortus btpA, B. abortus btpB, B. abortus btpAbtpB or B. abortus Bpe159 mutants. All infections were done in the presence of 150 U/ml IFN-γ (Endogen) for 2 h in standard medium containing no antibiotics. In another set of experiments, BMM from WT or TLR7 KO mice at a concentration of 0.5 x 106/ml were infected in a 24-well plate with different MOI of B. abortus S2308. Infections were done in the presence of 10 ng/ml mIFN-γ (Peprotech) for 2 h in standard medium containing no antibiotics. In all cases, cells were extensively washed to remove uninternalized bacteria and infected cells were maintained in culture in the presence of IFN-γ or mIFN-γ, 100 μg/ml gentamicin and 50 μg/ml streptomycin for an additional 48 h.THP-1 cells at a concentration of 0.5 x 106/ml were treated with different doses of B. abortus RNA in the presence of IFN-γ for 48 h. THP-1 cells treated with 2% paraformaldehyde (PFA) were also included as positive control. After 48 h, cells were washed and incubated with Annexin V-FITC and Propidium Iodide (BD Biosciences) for 10 min on ice in darkness. Then, cells were evaluated in the quadrants of Annexin V+/PI- (early apoptosis), Annexin V+/PI+ (late apoptosis) and Annexin V-/PI+ (necrosis). After labelling, cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using CellQuest software (BD Biosciences).For viability assay, THP-1 cells at a concentration of 0.5 x 106 PBMCs or 5 x 108 CFU were resuspended in 1ml of TRIzol Reagent (Invitrogen) and total RNA was extracted according to the manufacturer’s instructions. OD at 260 was measured for RNA quantification. In another set of experiments, B. abortus RNA was purified with Quick-RNA MiniPrep (Zymo Research) according to the manufacturer’s instructions. The purity of B. abortus RNA was assessed using a DeNovix DS-11 Spectrophotometer (DeNovix Inc.) with a ratio of absorbance 260/280 > 2.0 and a ratio of absorbance 260/230 > 1.8. In one set of experiments, RNA was treated with DNase RQ1 (Promega), Proteinase K (PK) (Promega) or E. coli RNase I (Life Technologies) prior to cell stimulation. RNA preparations  were further visualized by 1% agarose gel electrophoresis. The RNA was detected using UV light and the size of the RNA was determined using standard 100 bp Plus DNA ladder .5–10 x 106/ml were stimulated with B. abortus RNA, other prokaryotic or eukaryotic RNAs, DNase-treated B. abortus RNA, PK-treated B. abortus RNA, RNase I-treated B. abortus RNA, HK B. abortus strains, different structural components of B. abortus  or TLR ligands in the presence of 150 U/ml IFN-γ for 48 h in standard medium containing antibiotics. In another set of experiments, THP-1 cells were treated with B. abortus RNA complexed with Lipofectamine 2000 (Invitrogen). Briefly, Lipofectamine was mixed with bacterial RNA (1:3 ratio) in 100 μL/well serum-free RPMI and incubated for 20 min at room temperature. Then, complexes were added to the cells in the presence of 1.25% FBS and cell cultures were incubated for 48 h at 37°C in a 5% CO2 atmosphere. In all cases, MHC-I expression was evaluated by flow cytometry.Cells at 0.5 x 10B. abortus infection or stimulation; THP-1 cells or human primary monocytes were stained with FITC-labelled anti-human HLA-ABC  or isotype-matched control mAbs. In the experiments with murine macrophages, BMM were infected with B. abortus, or treated with B. abortus RNA or TLR ligands in the presence of 10 ng/ml recombinant murine IFN-γ (PeproTech) for 48 h. To determine MHC-I surface expression, cells were stained with PE- or FITC-labelled anti-mouse H-2Kd/H-2Dd . In all cases, cells were washed and incubated with 7-Amino-Actimycin D  for 10 min on ice in darkness. MHC-I expression was evaluated gating on viable cells (7-AAD negative cells). After labelling, cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using CellQuest software (BD Bioscience) or FlowJo 7.6 software.After 5 cells/well) with 10 ng/ml PMA (Sigma-Aldrich) for 24 h to promote adherence. Then, cells were infected with B. abortus or stimulated with B. abortus RNA or RNase I-treated B. abortus RNA in the presence of IFN-γ for 48 h, fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin and incubated with anti-HLA-ABC class I mAb W6/32, (purified from murine hybridoma culture supernatants) and Alexa 546-labelled secondary Ab (Invitrogen). Subcellular compartments were detected using mAbs specific for EEA1 (early endosomes), LAMP-2 (late endosomes/lysosomes), GM130 (Golgi) and calnexin (ER) (BD Biosciences) following Alexa 488-labelled secondary Ab (Invitrogen). Slides were mounted with PolyMount (Polysciences) and analyzed using FV-1000 confocal microscope with an oil-immersion Plan Apochromatic 60X NA1.42 objective (Olympus).THP-1 cells were incubated in chambers-slides (2 x 10b (SIINFEKL) was detected using the T cell hybridoma B3Z, which carries a β-galactosidase construct driven by NF-AT elements from the IL-2 promoter [B. abortus RNA-treated BMM from WT or TLR7 KO mice were exposed to 20 ng/ml of the SIINFEKL epitope during 20 min at 37°C. Then cells were washed, suspended in complete medium, and cultured in the presence of the T cell hybridoma B3Z. After 0, 4, 6 and 18 h of culture, cells were washed with PBS, and the cross-presentation was evaluated by a colorimetric assay using o-nitrophenyl-p-D-galactoside (ONPG) (Sigma-Aldrich) as substrate to detect the LacZ activity in B3Z lysates.Presentation of OVA epitope 257–264 on Kpromoter . For Ag Antibody targeting EGFR (Cetuximab) was purchased from Merck Serono. Gardiquimod, R848 (Resiquimod), ssRNA40/LyoVec and ORN06/LyoVec were purchased from InvivoGen. The 257–264 OVA peptide (SIINFEKL) was provided by Dr. S. Amigorena . TLR3/dsRNA Complex Inhibitor was purchased from Calbiochem.Human TNF-α and IL-1β were measured in culture supernatants by sandwich ELISA, using paired cytokine-specific mAbs according to the manufacturer’s instructions (BD Pharmingen).post hoc Tukey test using the GraphPad Prism software.Results were analyzed with one-way ANOVA followed by B. abortus is an intracellular pathogen capable of surviving inside macrophages [B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD8+ T cells responses that it elicits. Previously, we have demonstrated that B. abortus infection inhibits the IFN-γ-induced MHC-I surface expression on human monocytes down-modulating cytotoxic CD8+ T cell responses [B. abortus components involved. First of all, heat-killed B. abortus is incapable of inhibiting MHC-I expression [rophages . The sucesponses . Moreoveesponses . Two strpression . Secondlpression . TogetheBrucella employs to inhibit MHC-I on monocytes/macrophages is a component associated with bacterial viability itself regardless of the most relevant bacterial virulence factors. Particularly, we elucidated that this component is B. abortus RNA. Moreover, our experiments demonstrated that not only wild-type B. abortus RNA but also the RNA of two mutants strains, RB51 and virB10, were equally able to inhibit MHC-I. These results together with those shown in B. abortus to inhibit MHC-I surface expression is due to the absence of RNA in these bacterial preparations. In line with this evidence, it is widely known that live vaccines trigger more vigorous immune responses than their killed counterparts, even when live microorganisms are attenuated by elimination of their virulence factors [vita-PAMP present only in viable bacteria, triggering a unique innate and a robust adaptive antibody responses[et al in their study show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided they are alive [B. abortus as it could be extended to other prokaryotic RNAs, suggesting the broad implications of this immune regulation in the context of other infectious processes.In this study, we could corroborate that what  factors . Since sB. abortus infection, the human monocyte/macrophages are able to sense bacterial RNA. This question can be answered if we review the biology of B. abortus and the localization of receptors capable of detecting RNA. It has been described that viral and bacterial RNA are sensed by pattern recognition receptors (PRRs), among which the TLRs family has gained more attention [B. abortus is able to enter, survive and replicate within vacuolar phagocytic compartments of macrophages [Brucella dwells in an acidified compartment that fuses with components of the early and late endosomal/lysosomes pathway [Brucella-containing vacuole (BCV), which traffics from the endocytic compartment to the endoplasmic reticulum (ER) [et al demonstrated that Brucella replication in the ER is followed by BCV conversion into multi-membrane LAMP-1-positive vacuoles with autophagic features (aBCV). Furthermore, aBCVs were required to complete the intracellular Brucella lifecycle and for cell-to-cell spreading [B. abortus traffics through early and late endosomes/lysosomes the bacterial RNA released during phagocytosis activate endolysosomal TLRs. On the other hand, B. abortus mutant strains on virulence factors are also capable of infecting human monocytes/macrophages and transiting by early and late endosomes/lysosomes, but, unlike wild-type B. abortus, they are unable to replicate in BCVs and thus persist into the cell host. However, our results demonstrated that these strains are equally able to down-regulate MHC-I than wild-type B. abortus. As a consequence, the RNA of these bacteria could also gain access to TLR3, TLR7 or TLR8 in their transit through endosomes and lysosomes, although they do not persist in macrophages. In accordance with this, it was reported that human TLR8 is activated upon recognition of Borrelia burgdorferi RNA in the phagosome of human monocytes [in vitro experiments of stimulation with purified RNA, either in the presence or the absence of transfection, the entry of RNA by endocytosis gaining access to the endosomal TLRs can perfectly mimic what happens in an infectious context.One issue that merits discussion is how, during ttention , 27. TLRttention . With rerophages . Once in pathway . There, lum (ER) , 31. Oncpreading . In thisonocytes . Furtheronocytes . This goonocytes . In our B. abortus RNA. However, an hTLR7 agonist per se was unable to reproduce MHC-I inhibition. This led us to propose hTLR8 as a possible receptor, which was corroborated by the specific human TLR8 agonists ssRNA40 and ORN06. The greater efficiency of the synthetic oligonucleotide ORN06 in MHC-I inhibition may be due to the fact that it contains 6 repeats of the UUGU sequence motif, identified as the minimal motif responsible for ssRNA40 immunoactivity [B. abortus RNA-mediated MHC-I inhibition was corroborated in murine BMM using the agonist ssRNA40 which is also specific for murine TLR7, since TLR7 acts as the human TLR8 in mice [B. abortus and its RNA is mediated by hTLR8/mTLR7. In agreement with these results, the inhibition of antigen presentation to CD8+ T cells by B. abortus RNA was abolished in BMM from TLR7 KO mice. Further experiments in vivo are needed to determine the involvement of TLR8/TLR7 in the cytotoxic CD8+ T cell responses and chronicity of B. abortus-infected mice.Regarding the receptor involved, our results indicated that an hTLR7/8 agonist such as R848 was able to mimic the MHC-I inhibition mediated by activity . In addi in mice , 18. MorSingle-stranded RNA has been identified as the natural ligand of TLR7 and TLR8 , 35. Of et al have recently demonstrated that both receptors play an important role in sensing B. abortus RNA to induce the production of pro-inflammatory cytokines and type I IFN expression in murine DCs. However, these receptors were not required to control Brucella infection in vivo [B. abortus RNA sensing. However, we did not focus our attention on TLR13 since it is a receptor present in mice but not in humans [Listeria monocytogenes by sensing the RNA secreted into the cytosol of infected cells [B. abortus RNA, taking into account the cytosolic location of such receptors, the RNA should be able to be transferred from the phagosomes into the cell cytosol. However, this does not occur for all bacteria as it was demonstrated for B. burgdorferi RNA [Brucella, it was demonstrated that B. abortus DNA can activate cytosolic molecules such as AIM2 and STING [Although our results indicated that human TLR3 and TLR7 are not involved in MHC-I inhibition, Campos  in vivo . To expl in vivo , 41, coun humans , 41. Beyn humans , 43. Mored cells , 45. Altferi RNA . Regardind STING , 47; howB. abortus infection [We have previously demonstrated that the EGFR pathway is involved in the inhibition of MHC-I mediated by nfection . Moreovenfection . In lighnfection , we nextB. abortus induces the release of its RNA and RNA degradation products into the Brucella-containing endosomes. These molecules via TLR8 induce the secretion of EGF-like ligands such as EGF and TGF-α which bind ErbB receptors on the cell surface causing their activation. These effects finally lead to the retention of MHC-I molecules within the Golgi apparatus. MHC-I molecules are therefore unable to reach the cell surface and present bacterial Ags to CD8+ T cells  or virB10- (B) at different MOI in the presence of IFN-γ for 2 h, washed and cultured in the presence of IFN-γ for 48 h.  At the same time, heat-killed bacteria (HK) were used to treat THP-1 cells in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Bars represent the arithmetic means ± SEM of five experiments. MFI, mean fluorescence intensity. ***P<0.001 vs. IFN-γ-treated. THP-1 cells were infected with (TIF)Click here for additional data file.S2 FigB. abortus RNA purified with Quick-RNA MiniPrep kit in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Bars indicate the arithmetic means ± SEM of five independent experiments. MFI, mean fluorescence intensity. **P<0.01 vs. IFN-γ-treated.THP-1 cells were treated with different doses of (TIF)Click here for additional data file.S3 FigB. abortus or HKBA. In addition, B. abortus RNA was heat-treated at 70°C for 20 min. Each preparation was visualized by 1% agarose gel electrophoresis. (B) THP-1 cells were stimulated with B. abortus RNA, RNA extraction products from HKBA or heat-treated B. abortus RNA in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Bars represent the arithmetic means ± SEM of three experiments. MFI, mean fluorescence intensity. **P<0.01 vs. IFN-γ-treated.(A) RNA was purified from (TIF)Click here for additional data file.S4 FigB. abortus RNA in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Bars indicate the arithmetic means ± SEM of five independent experiments. MFI, mean fluorescence intensity. **P<0.01 vs. IFN-γ-treated.THP-1 cells were treated with HKBA with or without (TIF)Click here for additional data file.S5 FigB. abortus RNA (10 μg/ml) in the presence of IFN-γ and in the presence or absence of an endocytosis inhibitor (Nystatin) for 48 h. MHC-I expression was assessed by flow cytometry. Bars indicate the arithmetic means ± SEM of five independent experiments. MFI, mean fluorescence intensity. *P<0.05 vs. IFN-γ-treated; ##P<0.01 vs. Control.THP-1 cells were treated with (TIF)Click here for additional data file.S6 FigS. typhimurium (A), B. cereus (B), E. coli (C) or K. pneumoniae (D) RNAs in the presence of IFN-γ for 48 h. (E) THP-1 cells were treated with different doses of PBMCs RNA in the presence of IFN-γ for 48 h. MHC-I expression was assessed by flow cytometry. Bars indicate the arithmetic means ± SEM of three independent experiments. MFI, mean fluorescence intensity. *P<0.05; **P<0.01; ***P<0.001 vs. IFN-γ-treated.(A-D) THP-1 cells were treated with different doses of (TIF)Click here for additional data file.S7 FigB. abortus RNA in the presence of Cetuximab or Isotype control for 24 h. Supernatants were then collected and the amount of TNF-α (A) or IL-1β (B) was determined by ELISA. Bars indicate the arithmetic means ± SEM of three independent experiments. ***P<0.001 vs. untreated + Isotype or Cetuximab, accordingly.(A and B) THP-1 cells were treated with ORN06 or (TIF)Click here for additional data file.