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.

• 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

Tick-borne encephalitis virus (TBEV) is flavivirus transmitted to the host via tick saliva which contains various molecules with biological impacts. One of such molecules is Iristatin, a cysteine protease inhibitor from Ixodes ricinus that has been shown to have immunomodulatory properties. To characterize Iristatin in the relation to TBEV, we investigate whether this tick inhibitor has any capacity to influence TBEV infection. Mice were intradermally infected by TBEV with or without Iristatin and the viral multiplication was determined in skin and brain tissues by RT-PCR two and 5 days after infection. The viral RNA was detected in both intervals in skin and increased by time. The application of Iristatin caused a reduction in viral RNA in skin but not in the brain of infected mice 5 days post-infection. Moreover, anti-viral effect of Iristatin on skin was accompanied by a significant decline of interferon-stimulated gene 15 gene expression. The effect of Iristatin on TBEV replication was tested also in vitro in primary macrophages and dendritic cells; however, no changes were observed suggesting no direct interference of Iristatin with virus replication. Still, the Iristatin caused a suppression of Erk1/2 phosphorylation in TBEV-infected dendritic cells and had the anti-apoptotic effect. This is the first report showing that a tick cystatin decreases the viral RNA in the host skin, likely indirectly through creating skin environment that is less supportive for TBEV replication. Assuming, that viral RNA reflects the amount of infectious virus, decline of TBEV in host skin could influence the tick biology or virus transmission during cofeeding.

• MeSH
• antivirové látky farmakologie   MeSH
• cystatiny farmakologie   metabolismus   genetika   MeSH
• dendritické buňky virologie   účinky léků   MeSH
• klíště * virologie   účinky léků   MeSH
• klíšťová encefalitida * virologie   MeSH
• kůže * virologie   MeSH
• makrofágy virologie   MeSH
• mozek virologie   metabolismus   MeSH
• myši MeSH
• replikace viru * účinky léků   MeSH
• RNA virová genetika   MeSH
• slinné cystatiny metabolismus   MeSH
• viry klíšťové encefalitidy * účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• MeSH
• buněčné jádro metabolismus   virologie   MeSH
• DEAD-box RNA-helikasy metabolismus   genetika   MeSH
• genom virový MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus * genetika   metabolismus   fyziologie   MeSH
• replikace viru genetika   fyziologie   MeSH
• RNA virová * metabolismus   genetika   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• sestavení viru * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

• MeSH
• genom virový MeSH
• lidé MeSH
• počátek transkripce * MeSH
• promotorové oblasti (genetika) MeSH
• RNA virová genetika   MeSH
• sekvenční analýza RNA * metody   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• virus opičích neštovic genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the -1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• molekulární modely MeSH
• preklinické hodnocení léčiv * metody   MeSH
• RNA virová * metabolismus   antagonisté a inhibitory   genetika   MeSH
• SARS-CoV-2 * účinky léků   MeSH
• simulace molekulového dockingu MeSH
• uživatelské rozhraní počítače MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

• MeSH
• 3' nepřekládaná oblast * MeSH
• chybné párování bází * MeSH
• COVID-19 virologie   MeSH
• genom virový MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• párování bází MeSH
• Plasmodium falciparum genetika   MeSH
• RNA virová * chemie   genetika   MeSH
• SARS-CoV-2 * genetika   chemie   MeSH
• simulace molekulární dynamiky MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.

• MeSH
• COVID-19 * virologie   diagnóza   genetika   MeSH
• dospělí MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nazofarynx virologie   MeSH
• pitva * metody   MeSH
• plíce virologie   patologie   diagnostické zobrazování   MeSH
• RNA virová * genetika   analýza   MeSH
• SARS-CoV-2 * genetika   MeSH
• senioři MeSH
• testování na COVID-19 průkazem nukleové kyseliny metody   MeSH
• trachea virologie   patologie   diagnostické zobrazování   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.

• MeSH
• chřipka lidská virologie   MeSH
• elektronová kryomikroskopie * MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• replikace viru MeSH
• RNA virová metabolismus   genetika   MeSH
• RNA-dependentní RNA-polymerasa * metabolismus   chemie   MeSH
• RNA-polymerasa II * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• virové proteiny * metabolismus   chemie   genetika   MeSH
• virus chřipky A * metabolismus   genetika   enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Global impact of COVID-19 pandemic has heightened the urgency for efficient virus detection and identification of variants such as the Q57H mutation. Early and efficient detection of SARS-CoV-2 among densely populated developing countries is paramount objective. Although RT-PCR assays offer accuracy, however, dependence on expansive kits and availability of allied health resources pose an immense challenge for developing countries. In the current study, RT-LAMP based detection of SARS-Cov-2 with subsequent confirmation of Q57H variant through ARMS-PCR was performed. Among the 212 collected samples, 134 yielded positive results, while 78 tested negative using RT-LAMP. Oropharyngeal swabs of suspected individuals were collected and processed for viral RNA isolation. Isolated viral RNA was processed further by using either commercially available WarmStart Master Mix or our in house developed LAMP master mix separately. Subsequently, the end results of each specimen were evaluated by colorimetry. For LAMP assays, primers targeting three genes (ORF1ab, N and S) were designed using PrimerExplorer software. Interestingly, pooling of these three genes in single reaction tube increased sensitivity (95.5%) and specificity (93.5%) of LAMP assay. SARS-CoV-2 positive specimens were screened further for Q57H mutation using ARMS-PCR. Based on amplicon size variation, later confirmed by sequencing, our data showed 18.5% samples positive for Q57H mutation. Hence, these findings strongly advocate use of RT-LAMP-based assay for SARS-CoV-2 screening within suspected general population. Furthermore, ARMS-PCR also provides an efficient mean to detect prevalent mutations against SARS-Cov-2.

• MeSH
• COVID-19 * diagnóza   MeSH
• diagnostické techniky molekulární metody   MeSH
• lidé MeSH
• pandemie MeSH
• polymerázová řetězová reakce MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 * genetika   MeSH
• senzitivita a specificita MeSH
• testování na COVID-19 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.

• MeSH
• biosenzitivní techniky metody   MeSH
• DNA vazebné proteiny genetika   MeSH
• DNA virů genetika   MeSH
• elektrochemické techniky * metody   MeSH
• genom virový MeSH
• infekce papilomavirem * virologie   MeSH
• integrace viru * genetika   MeSH
• lidé MeSH
• lidský papilomavirus 16 * genetika   MeSH
• messenger RNA * genetika   MeSH
• nádory děložního čípku * virologie   MeSH
• onkogenní proteiny virové * genetika   MeSH
• Papillomavirus E7 - proteiny * genetika   MeSH
• progrese nemoci MeSH
• RNA virová genetika   MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH