Differentially-regulated miRNAs in COVID-19: A systematic review.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for coronavirus disease of 2019 (COVID-19) that infected more than 760 million people worldwide with over 6.8 million deaths to date. COVID-19 is one of the most challenging diseases of our times due to the nature of its spread, its effect on multiple organs, and an inability to predict disease prognosis, ranging from being completely asymptomatic to death. Upon infection, SARS-CoV-2 alters the host immune response by changing host-transcriptional machinery. MicroRNAs (miRNAs) are regarded as post-transcriptional regulators of gene expression that can be perturbed by invading viruses. Several in vitro and in vivo studies have reported such dysregulation of host miRNA expression upon SARS-CoV-2 infection. Some of this could occur as an anti-viral response of the host to the viral infection. Viruses themselves can counteract that response by mounting their own pro-viral response that facilitates virus infection, an aspect which may cause pathogenesis. Thus, miRNAs could serve as possible disease biomarkers in infected people. In the current review, we have summarised and analysed the existing data about miRNA dysregulation in patients infected with SARS-CoV-2 to determine their concordance between studies, and identified those that could serve as potential biomarkers during infection, disease progression, and death, even in people with other co-morbidities. Having such biomarkers can be vital in not only predicting COVID-19 prognosis, but also the development of novel miRNA-based anti-virals and therapeutics which can become invaluable in case of the emergence of new viral variants with pandemic potential in the future.

The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication Primarily Via Cluster Member miR-92a.

The mouse mammary tumor virus (MMTV) is a well-known causative agent of breast cancer in mice. Previously, we have shown that MMTV dysregulates expression of the host miR-17-92 cluster in MMTV-infected mammary glands and MMTV-induced tumors. This cluster, better known as oncomiR-1, is frequently dysregulated in cancers, particularly breast cancer. In this study, our aim was to uncover a functional interaction between MMTV and the cluster. Our results reveal that MMTV expression led to dysregulation of the cluster in both mammary epithelial HC11 and HEK293T cells with the expression of miR-92a cluster member being affected the most. Conversely, overexpression of the whole or partial cluster significantly repressed MMTV expression. Notably, overexpression of cluster member miR-92a alone repressed MMTV expression to the same extent as overexpression of the complete/partial cluster. Inhibition of miR-92a led to nearly a complete restoration of MMTV expression, while deletion/substitution of the miR-92a seed sequence rescued MMTV expression. Dual luciferase assays identified MMTV genomic RNA as the potential target of miR-92a. These results show that the miR-17-92 cluster acts as part of the cell's well-known miRNA-based anti-viral response to thwart incoming MMTV infection. Thus, this study provides the first evidence highlighting the biological significance of host miRNAs in regulating MMTV replication and potentially influencing tumorigenesis.

Global Down-regulation of Gene Expression Induced by Mouse Mammary Tumor Virus (MMTV) in Normal Mammary Epithelial Cells.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

Investigation of a herpesvirus outbreak in mixed breeds of adult domestic ducks using next generation sequencing.

This report characterizes the first lethal outbreak of Marek's disease on a large farm of mixed-breed adult ducks (>18,000) and identifies the pathogen that resulted in high mortality (35%). Clinical signs included inappetence, respiratory distress, depression, muscle weakness, and ataxia. Post mortem revealed enlarged fragile liver mottled with miliary whitish spots and an enlarged spleen. Histopathology revealed hepatocellular necrosis with eosinophilic intra-nuclear inclusion bodies, necrosis of splenic follicles and degeneration/necrosis of renal tubules. The disease was tentatively diagnosed as a herpesvirus infection, confirmed by virus isolation from the liver. DNA was isolated from 15-year-old archival formalin-fixed tissues from infected ducks and subjected to next generation sequencing (NGS). Despite highly degraded DNA, short stretches of G- and C-rich repeats (TTAGGG and TAACCC) were identified as telomeric repeats frequently found in herpesviruses. Megablast and further investigative bioinformatics identified presence of Marek's disease virus (MDV), a Gallid alphaherpesvirus type 2 (GAHV-2), as the cause of the acute fatal infection. The source of infection may be attributed to a dead migratory flamingo found close to the duck enclosures three days prior to the outbreak; hence, GAHV-2 may also be responsible for the fatal infection of the flamingo accentuated by heat stress. Considering the possible spread of this highly contagious and lethal virus from a flamingo to the ducks, and the increasing zoonosis of animal viruses into humans, such as monkey B alphaherpesvirus transmission from macaques to humans with ~80% fatality, this observation has important ramifications for human health and safety of the poultry industry.

Detection of SARS-CoV-2 in COVID-19 Patient Nasal Swab Samples Using Signal Processing.

This work presents an opto-electrical method that measures the viral nucleocapsid protein and anti-N antibody interactions to differentiate between SARS-CoV-2 negative and positive nasal swab samples. Upon light exposure of the patient nasal swab sample mixed with the anti-N antibody, charge transfer (CT) transitions within the altered protein folds are initiated between the charged amino acids side chain moieties and the peptide backbone that play the role of donor and acceptor groups. A Figure of Merit (FOM) was introduced to correlate the relative variations of the samples with and without antibody at two different voltages. Empirically, SARS-CoV-2 in patient nasal swab samples was detected within two minutes, if an extracted FOM threshold of >1 was achieved; otherwise, the sample wasconsidered negative.

MMTV does not encode viral microRNAs but alters the levels of cancer-associated host microRNAs.

Mouse mammary tumor virus (MMTV) induces breast cancer in mice in the absence of known virally-encoded oncogenes. Tumorigenesis by MMTV is thought to occur primarily through insertional mutagenesis, leading to the activation of cellular proto-oncogenes and outgrowth of selected cells. Here we investigated whether MMTV encodes microRNAs (miRNAs) and/or modulates host miRNAs that could contribute to tumorigenesis. High throughput small RNA sequencing analysis of MMTV-infected cells and MMTV-induced mammary tumors demonstrates that MMTV does not encode miRNAs. However, infected tissues have altered levels of several host miRNAs, including increased expression of members of the oncogenic miRNA cluster, miR-17-92. Notably, similar changes in miRNA levels have been previously reported in human breast cancers. Combined, our results demonstrate that virally encoded miRNAs do not contribute to MMTV-mediated tumorigenesis, but that changes in specific host miRNAs in infected cells may contribute to virus replication and tumor biology.

A cis-Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability.

BACKGROUND: The mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3' cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5' end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3' RmRE could facilitate translation of all other mRNAs, including gRNA. RESULTS: To address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. CONCLUSIONS: MMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.

Electrical detection and quantification of single and mixed DNA nucleotides in suspension.

High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

Electrical characterization of DNA supported on nitrocellulose membranes.

Integrated DNA-based nanoscale electronic devices will enable the continued realization of Moore's Law at the level of functional devices and systems. In this work, the electrical characterization of single and complementary base paired DNA has been directly measured and investigated via the use of nitrocellulose membranes. A radio frequency DAKS-3.5 was used to measure the reflection coefficients of different DNA solutions dotted onto nitrocellulose membranes. Each DNA solution was exposed to a radio frequency signal with a power of 10 dBm and with a sweep from 200 MHz up to 13.6 GHz. The conducted measurements show some distinctions between the homomeric and complementary bases due to their different electrical polarization. As revealed from the measurements conducted, with the addition of DNA oligonucleotides, the measured capacitance increased when compared with buffer medium alone. The DNA molecules could be modeled as dielectric material that can hold electrical charges. Furthermore, the complementary paired DNA molecule-based inks solutions had a higher capacitance value compared with single DNA molecules (A, C, G and T) solutions.