Neglected SARS-CoV-2 variants and potential concerns for molecular diagnostics: a framework for nucleic acid amplification test target site quality assurance.

IMPORTANCE: Molecular tests like polymerase chain reaction were widely used during the COVID-19 pandemic but as the pandemic evolved, so did SARS-CoV-2. This virus acquired mutations, prompting concerns that mutations could compromise molecular test results and be falsely negative. While some manufacturers may have in-house programs for monitoring mutations that could impact their assay performance, it is important to promptly report mutations in circulating viral strains that could adversely impact a diagnostic test result. However, commercial test target sites are proprietary, making independent monitoring difficult. In this study, SARS-CoV-2 test target sites were sequenced to monitor and assess mutations impact, and 29 novel mutations impacting SARS-CoV-2 detection were identified. This framework for molecular test target site quality assurance could be adapted to any molecular test, ensuring accurate diagnostic test results and disease diagnoses.

Hunting for mpox (monkeypox) mimickers: Use of the Biofire meningitis/encephalitis panel on lesion swabs to support alternative viral diagnoses.

BACKGROUND: Mpox (formerly monkeypox) is an emerging zoonotic disease of public health concern that presents as a rash mimicking other common viral exanthems. Unlike traditional testing algorithms relying on several assays, the BioFire FilmArray meningitis/encephalitis (ME) panel simultaneously detects common viruses causing rashes; however, Biofire ME is only licensed for testing on cerebral spinal fluid. OBJECTIVES: This study evaluated use of the Biofire ME panel for detection and discrimination of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV), human herpesviruses type 6 (HHV-6), enteroviruses (EVs), and human paraechoviruses (HPeVs) from a dermal or mucocutaneous swabs collected in universal transport media (UTM). STUDY DESIGN: Results of the BioFire ME panel were compared against methods used during clinical testing. Ten-fold serial dilutions in UTM of cultured viruses were used to compare analytical sensitivity, and analytical specificity was assessed using panels of microorganisms in UTM. Clinical sensitivity and specificity were assessed using 20 positive specimens each for HHV-1, HHV-2, HHV-6, VZV, EVs, and HPeV, as well as 35 known negative specimens that included 15 mpox-positive specimens. RESULTS: Biofire ME was as sensitive as comparator methods, and correctly discriminated all HSV-1, HSV-2, VZV, HHV-6, EVs, and HPeVs from mpox and mpox-mimickers. Cross-reaction between EV and rhinoviruses A, B, and C were noted in the specificity panel. CONCLUSIONS: Swabs in UTM collected for mpox testing are suitable for use on the Biofire ME panel, allowing more streamlined diagnostic testing for viral exanthems in patients under investigation for mpox infection.

Characterization of Swine Influenza A(H1N2) Variant, Alberta, Canada, 2020.

Influenza strains circulating among swine populations can cause outbreaks in humans. In October 2020, we detected a variant influenza A subtype H1N2 of swine origin in a person in Alberta, Canada. We initiated a public health, veterinary, and laboratory investigation to identify the source of the infection and determine whether it had spread. We identified the probable source as a local pig farm where a household contact of the index patient worked. Phylogenetic analysis revealed that the isolate closely resembled strains found at that farm in 2017. Retrospective and prospective surveillance using molecular testing did not identify any secondary cases among 1,532 persons tested in the surrounding area. Quick collaboration between human and veterinary public health practitioners in this case enabled a rapid response to a potential outbreak.

Molecular Pathogenicity of Enteroviruses Causing Neurological Disease.

Enteroviruses are single-stranded positive-sense RNA viruses that primarily cause self-limiting gastrointestinal or respiratory illness. In some cases, these viruses can invade the central nervous system, causing life-threatening neurological diseases including encephalitis, meningitis and acute flaccid paralysis (AFP). As we near the global eradication of poliovirus, formerly the major cause of AFP, the number of AFP cases have not diminished implying a non-poliovirus etiology. As the number of enteroviruses linked with neurological disease is expanding, of which many had previously little clinical significance, these viruses are becoming increasingly important to public health. Our current understanding of these non-polio enteroviruses is limited, especially with regards to their neurovirulence. Elucidating the molecular pathogenesis of these viruses is paramount for the development of effective therapeutic strategies. This review summarizes the clinical diseases associated with neurotropic enteroviruses and discusses recent advances in the understanding of viral invasion of the central nervous system, cell tropism and molecular pathogenesis as it correlates with host responses.

Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

The cell type resolved mouse transcriptome in neuron-enriched brain tissues from the hippocampus and cerebellum during prion disease.

Multiple cell types and complex connection networks are an intrinsic feature of brain tissue. In this study we used expression profiling of specific microscopic regions of heterogeneous tissue sections isolated by laser capture microdissection (LCM) to determine insights into the molecular basis of brain pathology in prion disease. Temporal profiles in two mouse models of prion disease, bovine spongiform encephalopathy (BSE) and a mouse-adapted strain of scrapie (RML) were performed in microdissected regions of the CA1 hippocampus and granular layer of the cerebellum which are both enriched in neuronal cell bodies. We noted that during clinical disease the number of activated microglia and astrocytes that occur in these areas are increased, thereby likely diluting the neuronal gene expression signature. We performed a comparative analysis with gene expression profiles determined from isolated populations of neurons, microglia and astrocytes to identify transcripts that are enriched in each of these cell types. Although the incubation periods of these two models are quite different, over 300 days for BSE and ~160 days for RML scrapie, these regional microdissections revealed broadly similar profiles. Microglial and astrocyte-enriched genes contributed a profound inflammatory profile consisting of inflammatory cytokines, genes related to phagocytosis, proteolysis and genes coding for extracellular matrix proteins. CA1 pyramidal neurons displayed a net upregulation of transcription factors and stress induced genes at pre-clinical stages of disease while all tissues showed profound decrease of overlapping genes related to neuronal function, in particular transcripts related to neuronal communication including glutamate receptors, phosphatase subunits and numerous synapse-related markers. Of note, we found a small number of genes expressed in neurons that were upregulated during clinical disease including, COX6A2, FZD9, RXRG and SOX11, that may be biomarkers of neurodegeneration.

Isolation of Viral-Infected Brain Regions for miRNA Profiling from Formalin-Fixed Paraffin-Embedded Tissues by Laser Capture Microdissection.

Brain is a highly heterogeneous organ with numerous layers of specialized cells. Viral infection further adds complexity to downstream analysis because only a subpopulation of the brain is infected. In these instances, molecular changes that occur within infected cells are not truly reflected when whole tissue is used for downstream analysis. Laser capture microdissection (LCM) is a tool that allows for the selection and isolation of cells or regions of interest as determined by microscopic observation. It provides a platform for visually selecting the tissue that truly represents the material one wishes to study, such as viral infected cells. Formalin-fixed paraffin-embedded viral-infected tissue allows for safe handling and processing by LCM. Here, we describe a method whereby viral-infected regions of the brain were specifically isolated by LCM from the rest of the FFPE tissue. The isolated regions were then used to extract RNA for microRNA profiling. This approach can be applied to study microRNA changes from any viral infection in any given tissue.

MicroRNA-16 targets mRNA involved in neurite extension and branching in hippocampal neurons during presymptomatic prion disease.

The mechanisms that lead to neuronal death in neurodegenerative diseases are poorly understood. Prion diseases, like many more common disorders such as Alzheimer's and Parkinson's diseases, are characterized by the progressive accumulation of misfolded disease-specific proteins. The earliest changes observed in brain tissue include a reduction in synaptic number and retraction of dendritic spines, followed by reduced length and branching of neurites. These pathologies are observable during presymptomatic stages of disease and are accompanied by altered expression of transcripts that include miRNAs. Here we report that miR-16 localized within hippocampal CA1 neurons is increased during early prion disease. Modulating miR-16 expression in mature murine hippocampal neurons by expression from a lentivirus, thus mimicking the modest increase seen in vivo, was found to induce neurodegeneration. This was characterized by retraction of neurites and reduced branching. We performed immunoprecipitation of the miR-16 enriched RISC complex, and identified associated transcripts from the co-immunoprecipitated RNA (Ago2 RIP-Chip). These transcripts were enriched with predicted binding sites for miR-16, including the validated miR-16 targets APP and BCL2, as well as numerous novel targets. In particular, genes within the neurotrophin receptor mediated MAPK/ERK pathway were potentially regulated by miR-16; including TrkB (NTRK2), MEK1 (MAP2K1) and c-Raf (RAF). Increased miR-16 expression in neurons during presymptomatic prion disease and reduction in proteins involved in MAPK/ERK signaling represents a possible mechanism by which neurite length and branching are decreased during early stages of disease.

MicroRNA and mRNA Dysregulation in Astrocytes Infected with Zika Virus.

The Zika virus (ZIKV) epidemic is an ongoing public health concern. ZIKV is a flavivirus reported to be associated with microcephaly, and recent work in animal models demonstrates the ability of the virus to cross the placenta and affect fetal brain development. Recent findings suggest that the virus preferentially infects neural stem cells and thereby deregulates gene expression, cell cycle progression, and increases cell death. However, neuronal stem cells are not the only brain cells that are susceptible to ZIKV and infection of other brain cells may contribute to disease progression. Herein, we characterized ZIKV replication in astrocytes, and profiled temporal changes in host microRNAs (miRNAs) and transcriptomes during infection. We observed the deregulation of numerous processes known to be involved in flavivirus infection, including genes involved in the unfolded protein response pathway. Moreover, a number of miRNAs were upregulated, including miR-30e-3p, miR-30e-5p, and, miR-17-5p, which have been associated with other flavivirus infections. This study highlights potential miRNAs that may be of importance in ZIKV pathogenesis.

DNA vaccination protects mice against Zika virus-induced damage to the testes.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Correction: Induction of Multiple miR-200/182 Members in the Brains of Mice Are Associated with Acute Herpes Simplex Virus 1 Encephalitis.

[This corrects the article DOI: 10.1371/journal.pone.0169081.].

Induction of Multiple miR-200/182 Members in the Brains of Mice Are Associated with Acute Herpes Simplex Virus 1 Encephalitis.

Important roles of microRNAs (miRNAs) in regulating the host response during viral infection have begun to be defined. However, little is known about the functional roles of miRNAs within an in vivo acute viral encephalitis model. We therefore identified global changes in miRNA expression during acute herpes simplex virus type 1 (HSV-1) encephalitis (HSVE) in mice. We found that many of the highly upregulated miRNAs (miR-155, miR-146a and miR-15b) detected in HSV-1 infected brain tissue are known regulators of inflammation and innate immunity. We also observed upregulation of 7 members belonging to the related group of miRNAs, the miR-200 family and miR-182 cluster (miR-200/182). Using in situ hybridization, we found that these miRNAs co-localized to regions of the brain with severe HSVE-related pathology and were upregulated in various cell types including neurons. Induction was apparent but not limited to cells in which HSV-1 was detected by immunohistochemistry, suggesting possible roles of these miRNAs in the host response to viral-induced tissue damage. Bioinformatic prediction combined with gene expression profiling revealed that the induced miR-200/182 members could regulate the biosynthesis of heparan sulfate proteoglycans. Using luciferase assays, we found that miR-96, miR-141, miR-183 and miR-200c all potentially targeted the syndecan-2 gene (Sdc2), which codes for a cell surface heparan sulfate proteoglycan involved in HSV-1 cellular attachment and entry.

Claudin 1 Expression Levels Affect miRNA Dynamics in Human Basal-Like Breast Cancer Cells.

Deemed a putative tumor suppressor in breast cancer, the tight junction protein claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype. Moreover, recent in vitro studies show that claudin 1 can regulate breast cancer cell motility and proliferation. Herein, we investigated whether microRNA (miRNA) dysregulation is associated with alterations in the level of claudin 1. Using next-generation sequencing (NGS), we identified seven miRNAs (miR-9-5p, miR-9-3p, let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) that were deregulated as a consequence of claudin 1 overexpression in the MDA-MB231 human breast cancer (HBC) cell line. Most of these miRNAs have been associated with tumor suppression in a variety of cancers, including breast cancer. Moreover, through gene expression profiling analysis, we identified epithelial-mesenchymal transition-related genes, including platelet-derived growth factor receptor-beta (PDGFRB) and cadherin 1 (CDH1, E cadherin), whose downregulation correlated with claudin 1 overexpression. Collectively, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Our data suggest that the dysregulated expression of these miRNAs, in conjunction with the high claudin 1 levels, could serve as a useful biomarker that identifies a subset of tumors within the poorly characterized basal-like subtype of breast cancer. Further studies are warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer.

MicroRNA abundance is altered in synaptoneurosomes during prion disease.

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

[Visual and anatomical outcomes of three intravitreal aflibercept injections in eyes with neovascular form of age-related macular degeneration]. / Anatomiczne i czynnosciowe wyniki podania trzech doszklistkowych iniekcji afliberceptu w oczach z neowaskularna postacia zwyrodnienia plamki zwiazanego z wiekiem.

PURPOSE: To evaluate results of treatment of choroidal neovascularization due to age-related macular degeneration with aflibercept in a group of patients representative for Polish population. MATERIAL AND METHODS: Three intravitreal injections of 2.0 mg of aflibercept were administrated to 48 eyes with active choroidal neovascularization at the four week intervals. In the evaluated population there were 19 eyes previously treated with other anti-VEGF drugs and 29 treatment naive eyes. RESULTS: The mean best corrected visual acuity at baseline in the entire group was 0.47. During the follow-up exam after the first injection, the best corrected visual acuity increased to 0.58. After the second and third injection, it reached the level of 0.62 and 0.63, respectively. Such improvement in visual acuity was accompanied by the reduction of central retinal thickness. Eyes with intraretinal fluid presence confirmed in baseline spectral domain optical coherence tomography had significantly lower best corrected visual acuity throughout the entire follow up. CONCLUSION: Three intravitreal aflibercept injections in eyes with neovascular age-related macular degeneration resulted in significant improvement of best corrected visual acuity and reduction of central retinal thickness both in eyes previously treated using other anti-VEGF drugs and newly diagnosed ones. aflibercept, neovascular age-related macular degeneration, choroidal neovascularization, anti-VEGF treatment, optical coherence tomography.

Activation of pro-survival CaMK4ß/CREB and pro-death MST1 signaling at early and late times during a mouse model of prion disease.

BACKGROUND: The signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo. METHODS: Mice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155-190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states. RESULTS: Hierarchical and functional clustering identified CaMK4ß and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4ß and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4ß/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi. CONCLUSION: Pro-survival CaMK4ß/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.

Microdissection and transcriptional profiling: a window into the pathobiology of preclinical prion disease.

Prion diseases share common features on a sub-cellular level with many neurodegenerative diseases including Alzheimer disease; the most prevalent neurodegenerative disease world-wide. The most obvious similarity is the accumulation of misfolded forms of the host proteins which forms aggregates in the brains of patients. Remarkably, one of the earliest pathological changes detected in degenerating brain tissue, well before clinical symptoms are observed, is synaptic dysfunction and loss. This pathology was recently shown to be reversible in early stages of mouse prion disease suggesting that synaptic regeneration and reestablishment of neuronal function is possible. Determination of the molecular events that underlie synapse degeneration and how this eventually results in neuronal loss is therefore a research priority that may contribute to the search for new therapeutic interventions for neurodegenerative disorders. Functional genomic studies using unbiased whole genome expression analyses represent one method that can provide insights into these perplexing processes. However, transcriptional profiles from brain tissues are representative of a heterogeneous mixture of cell types that effectively mask the expression of low abundance transcripts, or molecular changes that occur only in a small population of affected neurons. One method that was recently applied to address these challenges was laser capture microdissection which was used to effectively isolate the CA1 neuronal rich region of the hippocampus prior to RNA extraction. Profiling of both mRNAs and microRNAs revealed previously unidentified neuronal-specific genes and expression signatures that are relevant to understanding the pathophysiological processes involved in preclinical stages of prion disease. In this review we will highlight these molecular signatures and discuss their implications with respect to prion-induced neurodegeneration.

Small RNA drugs for prion disease: a new frontier.

INTRODUCTION: Prion diseases, also known as transmissible spongiform encephalopathies, are a group of neurodegenerative diseases that are invariably incurable. In fact, intense laboratory and clinical research have failed to discover effective treatments, to date, which delay the onset or progression of any neurodegenerative conditions, including those caused by infectious prions. It has become clear that profound changes in the brains of patients are evident long before clinical signs and it is at this stage that the disease is reversible and presents 'druggable' targets. However, research is beginning to uncover the molecular underpinnings involved in the early stages of disease pathogenesis. Targeting key genes and pathways using short non-coding RNA is a new avenue of exploratory research for the treatment of prion disease that holds much promise for the future. AREAS COVERED: This article reviews the novel approach of using RNA-based drugs as a therapeutic opportunity for prion disease. Furthermore, it discusses the challenges that currently exist in the development of these therapies and highlights the future opportunities in this area. EXPERT OPINION: Numerous challenges exist before this therapeutic option can be translated into effective treatments. First, the crucial genes and pathways targeted must be identified from the multitude of temporally and spatially altered genetic processes that occur during the disease. Second, patients must be before irreversible neuronal degeneration, that accompanies prion replication, has progressed. Finally, these small RNAs must be delivered to the affected region of the brain over long periods of time and without significant side effects.

The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: twintrons, introns, and intron-encoded proteins.

Comparative sequence analysis of the mitochondrial small subunit ribosomal RNA (rns) gene among species of Ophiostoma, Grosmannia, Ceratocystiopsis and related taxa provides an overview of the types of introns that have invaded this gene within the ophiostomatoid fungi. The rns gene appears to be a reservoir for a number of group I and group II introns along with intron-associated open reading frames such as homing endonucleases and reverse transcriptases. This study uncovered two twintrons, one at position mS917 where a group ID intron encoding a LAGLIDADG ORF invaded another ORF-less group ID intron. Another twintron complex was detected at position mS1247 here a group IIA1 intron invaded the open reading frame embedded within a group IC2 intron. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent losses. Results of this study will make a significant contribution to the understanding of the complexity of the mitochondrial intron landscape, and offer a resource to those annotating mitochondrial genomes. It will also serve as a resource to those that bioprospect for ribozymes and homing endonucleases.

A user-friendly computational workflow for the analysis of microRNA deep sequencing data.

Second-generation high-throughput sequencing is a robust and inexpensive methodology that is becoming an increasingly common technique for the study of microRNA (miRNA) expression levels in the central nervous system. This method allows for the identification of both known and novel miRNAs, reporting on the qualitative and quantitative levels these RNA species represent in any given sample. Numerous bioinformatic programs are currently available to analyze deep sequencing data but many require at least a partial understanding of the command line interface. In this chapter, we describe a user-friendly computational workflow guiding the user through the process from the initial FASTQ deep sequencing file to the identification of known and potentially novel miRNAs in a given experiment, as well as the assessment of the differential expression of these miRNAs between experimental samples. Furthermore, programs that can predict potential targets for these miRNAs are also highlighted.