Due to the fast global spreading of the Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2), prevention and treatment options are direly needed in order to control infection-related morbidity, mortality, and economic losses. Although drug and inactivated and attenuated virus vaccine development can require significant amounts of time and resources, DNA and RNA vaccines offer a quick, simple, and cheap treatment alternative, even when produced on a large scale. The spike protein, which has been shown as the most antigenic SARS-CoV-2 protein, has been widely selected as the target of choice for DNA/RNA vaccines. Vaccination campaigns have reported high vaccination rates and protection, but numerous unintended effects, ranging from muscle pain to death, have led to concerns about the safety of RNA/DNA vaccines. In parallel to these studies, several open reading frames (ORFs) have been found to be overlapping SARS-CoV-2 accessory genes, two of which, ORF2b and ORF-Sh, overlap the spike protein sequence. Thus, the presence of these, and potentially other ORFs on SARS-CoV-2 DNA/RNA vaccines, could lead to the translation of undesired proteins during vaccination. Herein, we discuss the translation of overlapping genes in connection with DNA/RNA vaccines. Two mRNA vaccine spike protein sequences, which have been made publicly-available, were compared to the wild-type sequence in order to uncover possible differences in putative overlapping ORFs. Notably, the Moderna mRNA-1273 vaccine sequence is predicted to contain no frameshifted ORFs on the positive sense strand, which highlights the utility of codon optimization in DNA/RNA vaccine design to remove undesired overlapping ORFs. Since little information is available on ORF2b or ORF-Sh, we use structural bioinformatics techniques to investigate the structure-function relationship of these proteins. The presence of putative ORFs on DNA/RNA vaccine candidates implies that overlapping genes may contribute to the translation of smaller peptides, potentially leading to unintended clinical outcomes, and that the protein-coding potential of DNA/RNA vaccines should be rigorously examined prior to administration.

• Klíčová slova
• DNA vaccine, ORF-Sh, ORF2b, RNA vaccine, SARS-CoV-2, codon optimization, spike protein,
• MeSH
• DNA vakcíny škodlivé účinky   genetika   MeSH
• glykoprotein S, koronavirus genetika   MeSH
• kodon MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• messenger RNA MeSH
• mRNA vakcíny škodlivé účinky   genetika   MeSH
• otevřené čtecí rámce MeSH
• překrývající se geny * MeSH
• proteinové domény MeSH
• proteosyntéza MeSH
• vakcíny proti COVID-19 škodlivé účinky   genetika   MeSH
• virové geny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vakcíny MeSH
• glykoprotein S, koronavirus MeSH
• kodon MeSH
• messenger RNA MeSH
• mRNA vakcíny MeSH
• spike protein, SARS-CoV-2 MeSH Prohlížeč
• vakcíny proti COVID-19 MeSH

• MeSH
• COVID-19 * prevence a kontrola   MeSH
• glykoprotein S, koronavirus MeSH
• lidé MeSH
• messenger RNA MeSH
• plíce MeSH
• příjemce transplantátu * MeSH
• protilátky virové MeSH
• SARS-CoV-2 MeSH
• T-lymfocyty MeSH
• vakcína BNT162 MeSH
• vakcíny proti COVID-19 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykoprotein S, koronavirus MeSH
• messenger RNA MeSH
• protilátky virové MeSH
• vakcína BNT162 MeSH
• vakcíny proti COVID-19 MeSH

Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on blood-derived fluids, other biofluids may be more informative. We present an atlas of messenger, circular, and small RNA transcriptomes of a comprehensive collection of 20 human biofluids. By means of synthetic spike-in controls, we compare RNA content across biofluids, revealing a 10,000-fold difference in concentration. The circular RNA fraction is increased in most biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. Our atlas enables an informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Spike-normalized data are publicly available in the R2 web portal for further exploration.

• Klíčová slova
• RNA sequencing, biofluids, biomarker, cell-free RNA, circular RNA, extracellular RNA, liquid biopsy, messenger RNA, small RNA,
• MeSH
• biologické markery * MeSH
• kohortové studie MeSH
• lidé MeSH
• RNA genetika   metabolismus   MeSH
• sekvenční analýza RNA metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• tělesné tekutiny metabolismus   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• biologické markery * MeSH
• RNA MeSH

UNLABELLED: We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. IMPORTANCE: The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full-genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material, and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes, including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We reanalyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic Middle East respiratory syndrome (MERS) coronavirus.

• MeSH
• Bayesova věta MeSH
• biologická evoluce * MeSH
• Chiroptera virologie   MeSH
• DNA primery genetika   MeSH
• feces virologie   MeSH
• fylogeneze * MeSH
• genetická variace * MeSH
• glykoprotein S, koronavirus genetika   MeSH
• lamy virologie   MeSH
• lidé MeSH
• lidský koronavirus 229E genetika   MeSH
• modely genetické MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Ghana MeSH
• Názvy látek
• DNA primery MeSH
• glykoprotein S, koronavirus MeSH
• RNA-dependentní RNA-polymerasa MeSH

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an RNA virus responsible for coronavirus disease 2019 (COVID-19). While SARS-CoV-2 primarily targets the lungs and airways, it can also infect other organs, including the central nervous system (CNS). The aim of this study was to investigate whether the choroid plexus could serve as a potential entry site for SARS-CoV-2 into the brain. Tissue samples from 24 deceased COVID-19-positive individuals were analyzed. Reverse transcription real-time PCR (RT-qPCR) was performed on selected brain regions, including the choroid plexus, to detect SARS-CoV-2 viral RNA. Additionally, immunofluorescence staining and confocal microscopy were used to detect and localize two characteristic proteins of SARS-CoV-2: the spike protein S1 and the nucleocapsid protein. RT-qPCR analysis confirmed the presence of SARS-CoV-2 viral RNA in the choroid plexus. Immunohistochemical staining revealed viral particles localized in the epithelial cells of the choroid plexus, with the spike protein S1 detected in the late endosomes. Our findings suggest that the blood-cerebrospinal fluid (B-CSF) barrier in the choroid plexus serves as a route of entry for SARS-CoV-2 into the CNS. This study contributes to the understanding of the mechanisms underlying CNS involvement in COVID-19 and highlights the importance of further research to explore potential therapeutic strategies targeting this entry pathway.

• Klíčová slova
• COVID‐19, SARS‐CoV‐2, blood‐cerebrospinal fluid barrier, choroid plexus, neuroinvasion,
• MeSH
• COVID-19 * virologie   MeSH
• dospělí MeSH
• fosfoproteiny * metabolismus   MeSH
• glykoprotein S, koronavirus * genetika   metabolismus   MeSH
• hematoencefalická bariéra * virologie   MeSH
• internalizace viru MeSH
• koronavirové nukleokapsidové proteiny MeSH
• lidé středního věku MeSH
• lidé MeSH
• plexus chorioideus * virologie   MeSH
• RNA virová * genetika   MeSH
• SARS-CoV-2 * fyziologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfoproteiny * MeSH
• glykoprotein S, koronavirus * MeSH
• koronavirové nukleokapsidové proteiny MeSH
• nucleocapsid phosphoprotein, SARS-CoV-2 MeSH Prohlížeč
• RNA virová * MeSH
• spike protein, SARS-CoV-2 MeSH Prohlížeč

The first outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in Wuhan, Hubei Province, China, in late 2019. The subsequent COVID-19 pandemic rapidly affected the health and economy of the world. The global approach to the pandemic was to isolate populations to reduce the spread of this deadly virus while vaccines began to be developed. In March 2020, the first phase I clinical trial of a novel lipid nanoparticle (LNP)-encapsulated mRNA-based vaccine, mRNA-1273, which encodes the spike protein (S protein) of SARS-CoV-2, began in the United States (US). The production of mRNA-based vaccines is a promising recent development in the production of vaccines. However, there remain significant challenges in the development and testing of vaccines as rapidly as possible to control COVID-19, which requires international collaboration. This review aims to describe the background to the rationale for the development of mRNA-based SARS-CoV-2 vaccines and the current status of the mRNA-1273 vaccine.

• MeSH
• Betacoronavirus * imunologie   MeSH
• COVID-19 MeSH
• glykoprotein S, koronavirus genetika   imunologie   MeSH
• koronavirové infekce imunologie   prevence a kontrola   MeSH
• lidé MeSH
• messenger RNA imunologie   MeSH
• pandemie prevence a kontrola   MeSH
• RNA virová imunologie   MeSH
• SARS-CoV-2 MeSH
• vakcína firmy Moderna proti COVID-19 MeSH
• vakcíny proti COVID-19 MeSH
• virová pneumonie imunologie   prevence a kontrola   MeSH
• virové vakcíny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Geografické názvy
• Spojené státy americké MeSH
• Názvy látek
• glykoprotein S, koronavirus MeSH
• messenger RNA MeSH
• RNA virová MeSH
• spike protein, SARS-CoV-2 MeSH Prohlížeč
• vakcína firmy Moderna proti COVID-19 MeSH
• vakcíny proti COVID-19 MeSH
• virové vakcíny * MeSH

Non-B nucleic acids structures have arisen as key contributors to genetic variation in SARS-CoV-2. Herein, we investigated the presence of defining spike protein mutations falling within inverted repeats (IRs) for 18 SARS-CoV-2 variants, discussed the potential roles of G-quadruplexes (G4s) in SARS-CoV-2 biology, and identified potential pseudoknots within the SARS-CoV-2 genome. Surprisingly, there was a large variation in the number of defining spike protein mutations arising within IRs between variants and these were more likely to occur in the stem region of the predicted hairpin stem-loop secondary structure. Notably, mutations implicated in ACE2 binding and propagation (e.g., ΔH69/V70, N501Y, and D614G) were likely to occur within IRs, whilst mutations involved in antibody neutralization and reduced vaccine efficacy (e.g., T19R, ΔE156, ΔF157, R158G, and G446S) were rarely found within IRs. We also predicted that RNA pseudoknots could predominantly be found within, or next to, 29 mutations found in the SARS-CoV-2 spike protein. Finally, the Omicron variants BA.2, BA.4, BA.5, BA.2.12.1, and BA.2.75 appear to have lost two of the predicted G4-forming sequences found in other variants. These were found in nsp2 and the sequence complementary to the conserved stem-loop II-like motif (S2M) in the 3' untranslated region (UTR). Taken together, non-B nucleic acids structures likely play an integral role in SARS-CoV-2 evolution and genetic diversity.

• Klíčová slova
• G-quadruplex, SARS-CoV-2, adaptation, inverted repeats, mutation, pseudoknot, spike protein,
• MeSH
• 3' nepřekládaná oblast MeSH
• COVID-19 * genetika   MeSH
• genomika MeSH
• glykoprotein S, koronavirus genetika   MeSH
• lidé MeSH
• nukleové kyseliny * MeSH
• SARS-CoV-2 genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• glykoprotein S, koronavirus MeSH
• nukleové kyseliny * MeSH
• spike protein, SARS-CoV-2 MeSH Prohlížeč

BACKGROUND: RT-qPCR quantification of miRNAs expression may play an essential role in pancreatic ductal adenocarcinoma (PDAC) diagnostics. RT-qPCR-based experiments require endogenous controls for the result normalization and reliability. However, expression instability of reference genes in tumors may introduce bias when determining miRNA levels. METHODS: We investigated expression of 6 miRNAs, isolated from FFPE samples of pancreatic adenocarcinomas. Four internal controls were utilized for RT-qPCR result normalization: artificial miR-39 from C. elegans, U6 snRNA, miR-16 and snoRNA U91. RESULTS: We found miR-21, miR-155 or miR-217 expression values in tumors may differ up to several times, depending on selected internal controls. Moreover, different internal controls can produce controversial results for miR-96, miR-148a or miR-196a quantification. Also, expression of our endogenous controls varied significantly in tumors. U6 demonstrated variation from -1.03 to 8.12-fold, miR-16 from -2.94 up to 7.38-fold and the U91 from -3.05 to 4.36-fold respectively. On the other hand, the most stable gene, determined by NormFinder algorithm, was U91. Each miRNA normalized relatively to the spike or U91, demonstrated similar expression values. Thus, statistically significant and insignificant differences between tumors and normal tissues for miRNAs were equal for the spike and the U91. Also, the differences between the spike and U91 were statistically insignificant for all of miRs except miR-217. Among three endogenous controls, U91 had the lowest average expression values and standard deviation in cancer tissues. CONCLUSIONS: We recommend U91 as a new normalizer for miRNA quantification in PDACs.

• MeSH
• adenokarcinom genetika   MeSH
• analýza rozptylu MeSH
• dospělí MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• malá jadérková RNA metabolismus   MeSH
• mikro RNA analýza   metabolismus   MeSH
• nádorové biomarkery genetika   MeSH
• nádory slinivky břišní genetika   MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá jadérková RNA MeSH
• mikro RNA MeSH
• MIRN155 microRNA, human MeSH Prohlížeč
• MIRN21 microRNA, human MeSH Prohlížeč
• MIRN217 microRNA, human MeSH Prohlížeč
• nádorové biomarkery MeSH

The pulsatile nature of gene activity has recently emerged as a general property of the transcriptional process. It has been shown that the frequency and amplitude of transcriptional bursts can be subjected to extrinsic regulation. Here we have investigated if these parameters were constant throughout the cell cycle using the single molecule RNA FISH technique. We found evidence of transcriptional spikes upon mitotic exit in three different human cell lines. Recording of cell growth prior to hybridization and immuno-RNA FISH analysis revealed that these spikes were short-lived and subsided before completion of cytokinesis. The transient post-mitotic increase in transcriptional output was found to be the result of cells displaying a higher number of active alleles and/or an increased number of nascent transcripts per active allele, indicating that both the burst fraction and the amplitude of individual bursts can be increased upon mitotic exit. Our results further suggest that distinct regulatory mechanisms are at work shortly after mitotic exit and during the rest of interphase. We speculate that transcriptional spikes are associated with chromatin decondensation, a hallmark of post-mitotic cells that might alter the dynamics of transcriptional regulators and effectors.

• MeSH
• alely MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• buňky Hep G2 MeSH
• CD antigeny genetika   izolace a purifikace   MeSH
• DNA řízené RNA-polymerasy genetika   izolace a purifikace   MeSH
• genetická transkripce * MeSH
• hybridizace in situ fluorescenční metody   MeSH
• lidé MeSH
• mitóza genetika   MeSH
• receptory transferinu genetika   izolace a purifikace   MeSH
• RNA genetika   MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH
• CD71 antigen MeSH Prohlížeč
• DNA řízené RNA-polymerasy MeSH
• POLR2A RNA polymerase, human MeSH Prohlížeč
• receptory transferinu MeSH
• RNA MeSH

The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.

• Klíčová slova
• CpG depletion, SARS-CoV-2, coronavirus, cytosine deamination, evolution, mutation bias,
• MeSH
• Betacoronavirus klasifikace   genetika   izolace a purifikace   MeSH
• Chiroptera virologie   MeSH
• CpG ostrůvky MeSH
• cytosin metabolismus   MeSH
• fylogeneze MeSH
• glykoprotein S, koronavirus genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• SARS-CoV-2 MeSH
• sekvence nukleotidů MeSH
• uracil metabolismus   MeSH
• virus SARS klasifikace   genetika   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytosin MeSH
• glykoprotein S, koronavirus MeSH
• uracil MeSH