FASTKD1 and FASTKD4 have opposite effects on expression of specific mitochondrial RNAs, depending upon their endonuclease-like RAP domain.

FASTK family proteins have been identified as regulators of mitochondrial RNA homeostasis linked to mitochondrial diseases, but much remains unknown about these proteins. We show that CRISPR-mediated disruption of FASTKD1 increases ND3 mRNA level, while disruption of FASTKD4 reduces the level of ND3 and of other mature mRNAs including ND5 and CYB, and causes accumulation of ND5-CYB precursor RNA. Disrupting both FASTKD1 and FASTKD4 in the same cell results in decreased ND3 mRNA similar to the effect of depleting FASTKD4 alone, indicating that FASTKD4 loss is epistatic. Interestingly, very low levels of FASTKD4 are sufficient to prevent ND3 loss and ND5-CYB precursor accumulation, suggesting that FASTKD4 may act catalytically. Furthermore, structural modeling predicts that each RAP domain of FASTK proteins contains a nuclease fold with a conserved aspartate residue at the putative active site. Accordingly, mutation of this residue in FASTKD4 abolishes its function. Experiments with FASTK chimeras indicate that the RAP domain is essential for the function of the FASTK proteins, while the region upstream determines RNA targeting and protein localization. In conclusion, this paper identifies new aspects of FASTK protein biology and suggests that the RAP domain function depends on an intrinsic nucleolytic activity.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression.

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

Orthopox viruses: is the threat growing?

BACKGROUND: Smallpox was a major cause of human mortality until its eradication, but the threat of orthopox viruses has not disappeared. Since the eradication of smallpox and the cessation of the related vaccination campaigns, the threat has been growing, as evidenced by the currently ongoing worldwide Mpox outbreak. In addition to threats of an evolving Mpox, we must also be aware of a myriad of other threats that remain. Many countries still lack biosecurity regulations reflecting the recent technological advances, and the threat of bioterrorism remains ever present. Reconstruction of smallpox is a distinct possibility, as are other scenarios whereby other orthopox viruses may be made more fit for transmission in humans. OBJECTIVES: To outline and discuss potential biosafety and biosecurity threats posed by orthopox viruses. SOURCES: Published scientific literature, news articles, and international agreements. CONTENT AND IMPLICATIONS: It would be wise to take steps to mitigate these threats now. Vaccination campaigns should be considered in areas with frequent orthopox outbreaks, and more efforts must be made to put a final end to the Mpox outbreak. In many countries, national biosafety and biosecurity regulations may need to be revised and strengthened to better reflect the threats posed by new technologies, including controls on synthesis of smallpox sequences. Furthermore, more international cooperation and aid is needed. The present global Mpox outbreak could likely have been prevented had areas where Mpox is endemic not been neglected. Future outbreaks could be much worse.

Novel SARS-CoV-2 variants: the pandemics within the pandemic.

BACKGROUND: Many new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been termed variants of concern/interest (VOC/I) because of the greater risk they pose due to possible enhanced transmissibility and/or severity, immune escape, diagnostic and/or treatment failure, and reduced vaccine efficacy. AIMS: We sought to review the current knowledge of emerging SARS-CoV-2 variants, particularly those deemed VOC/Is: B.1.351, B.1.1.7, and P.1. SOURCES: MEDLINE and BioRxiv databases, as well as the grey literature, were searched for reports of SARS-CoV-2 variants since November 2020. Relevant articles and their references were screened. CONTENT: Mutations on the spike protein in particular may affect both affinity for the SARS-CoV-2 cell receptor ACEII and antibody binding. These VOC/Is often share similar mutation sets. The N501Y mutation is shared by the three main VOCs: B.1.1.7, first identified in the United Kingdom, P.1, originating from Brazil, and B.1.351, first described in South Africa. This mutation likely increases transmissibility by increasing affinity for ACEII. The B.1.351 and P.1 variants also display the E484K mutation which decreases binding of neutralizing antibodies, leading to partial immune escape; this favours reinfections, and decreases the in vitro efficacy of some antibody therapies or vaccines. Those mutations may also have phenotypical repercussions of greater severity. Furthermore, the accumulation of mutations poses a diagnostic risk (lowered when using multiplex assays), as seen for some assays targeting the S gene. With ongoing surveillance, many new VOC/Is have been identified. The emergence of the E484K mutation independently in different parts of the globe may reflect the adaptation of SARS-CoV-2 to humans against a background of increasing immunity. IMPLICATIONS: These VOC/Is are increasing in frequency globally and pose challenges to any herd immunity approach to managing the pandemic. While vaccination is ongoing, vaccine updates may be prudent. The virus continues to adapt to transmission in humans, and further divergence from the initial Wuhan sequences is expected.

Analytical Evaluation of Visby Medical RT-PCR Portable Device for Rapid Detection of SARS-CoV-2.

Extended community testing constitutes one of the main strategic pillars in controlling the COVID-19 pandemic. Reverse transcription PCR (RT-PCR) targeting the SARS-CoV-2 genome on nasopharyngeal swab samples is currently the reference test. While displaying excellent analytical sensitivity and specificity, this test is costly, often requires a substantial turnaround time, and, more importantly, is subject to reagent and other material shortages. To complement this technology, rapid antigen tests have been developed and made available worldwide, allowing cheap, quick, and decentralized SARS-CoV-2 testing. The main drawback of these tests is the reduced sensitivity when RT-PCR is the gold standard. In this study, we evaluate Visby an innovative, portable, easy-to-use RT-PCR point-of-care (POC) diagnostic device. Our retrospective analysis shows that overall, compared to the Cobas 6800 RT-qPCR assay (Roche), this RT-PCR POC technology detects SARS-CoV-2 RNA with 95% sensitivity (95%CI = 86.3-99%) and 100% specificity (95% CI = 80.5-100%). For samples with cycle-threshold values below 31, we observed 100% sensitivity (95% CI = 66.4-100%). While showing an analytical sensitivity slightly below that of a standard RT-qPCR system, the evaluated Visby RT-PCR POC device may prove to be an interesting diagnostic alternative in the COVID-19 pandemic, potentially combining the practical advantages of rapid antigen tests and the robust analytical performances of nucleic acid detection systems.

Diagnostic accuracy of Panbio rapid antigen tests on oropharyngeal swabs for detection of SARS-CoV-2.

BACKGROUND: Antigen-detecting rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 offer new opportunities for testing in the context of the COVID-19 pandemic. Nasopharyngeal swabs (NPS) are the reference sample type, but oropharyngeal swabs (OPS) may be a more acceptable sample type in some patients. METHODS: We conducted a prospective study in a single screening center to assess the diagnostic performance of the Panbio™ COVID-19 Ag Rapid Test (Abbott) on OPS compared with reverse-transcription quantitative PCR (RT-qPCR) using NPS during the second pandemic wave in Switzerland. RESULTS: 402 outpatients were enrolled in a COVID-19 screening center, of whom 168 (41.8%) had a positive RT-qPCR test. The oropharyngeal Ag-RDT clinical sensitivity compared to nasopharyngeal RT-qPCR was 81% (95%CI: 74.2-86.6). Two false positives were noted out of the 234 RT-qPCR negative individuals, which resulted in a clinical specificity of 99.1% (95%CI: 96.9-99.9) for the Ag-RDT. For cycle threshold values ≤ 26.7 (≥ 1E6 SARS-CoV-2 genomes copies/mL, a presumed cut-off for infectious virus), 96.3% sensitivity (95%CI: 90.7-99.0%) was obtained with the Ag-RDT using OPS. INTERPRETATION: Based on our findings, the diagnostic performance of the Panbio™ Covid-19 RDT with OPS samples, if taken by a trained person and high requirements regarding quality of the specimen, meet the criteria required by the WHO for Ag-RDTs (sensitivity ≥80% and specificity ≥97%) in a high incidence setting in symptomatic individuals.

Diagnostic accuracy of two commercial SARS-CoV-2 antigen-detecting rapid tests at the point of care in community-based testing centers.

OBJECTIVES: Determine the diagnostic accuracy of two antigen-detecting rapid diagnostic tests (Ag-RDT) for SARS-CoV-2 at the point of care and define individuals' characteristics providing best performance. METHODS: We performed a prospective, single-center, point of care validation of two Ag-RDT in comparison to RT-PCR on nasopharyngeal swabs. RESULTS: Between October 9th and 23rd, 2020, 1064 participants were enrolled. The PanbioTM Covid-19 Ag Rapid Test device (Abbott) was validated in 535 participants, with 106 positive Ag-RDT results out of 124 positive RT-PCR individuals, yielding a sensitivity of 85.5% (95% CI: 78.0-91.2). Specificity was 100.0% (95% CI: 99.1-100) in 411 RT-PCR negative individuals. The Standard Q Ag-RDT (SD Biosensor, Roche) was validated in 529 participants, with 170 positive Ag-RDT results out of 191 positive RT-PCR individuals, yielding a sensitivity of 89.0% (95%CI: 83.7-93.1). One false positive result was obtained in 338 RT-PCR negative individuals, yielding a specificity of 99.7% (95%CI: 98.4-100). For individuals presenting with fever 1-5 days post symptom onset, combined Ag-RDT sensitivity was above 95%. Lower sensitivity of 88.2% was seen on the same day of symptom development (day 0). CONCLUSIONS: We provide an independent validation of two widely available commercial Ag-RDTs, both meeting WHO criteria of ≥80% sensitivity and ≥97% specificity. Although less sensitive than RT-PCR, these assays could be beneficial due to their rapid results, ease of use, and independence from existing laboratory structures. Testing criteria focusing on patients with typical symptoms in their early symptomatic period onset could further increase diagnostic value.

Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression.