Cross-reactivity and inclusivity analysis of CRISPR-based diagnostic assays of coronavirus SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named as 2019-nCoV) is the cause of the novel coronavirus disease 2019 (COVID-19) pandemic. Its diagnosis relies on the molecular detection of the viral RNA by polymerase chain reaction (PCR) while newer rapid CRISPR-based diagnostic tools are being developed. As molecular diagnostic assays rely on the detection of unique sequences of viral nucleic acid, the target regions must be common to all coronavirus SARS-CoV-2 circulating strains, yet unique to SARS-CoV-2 with no cross-reactivity with the genome of the host and other normal or pathogenic organisms potentially present in the patient samples. This stage 1 protocol proposes in silico cross-reactivity and inclusivity analysis of the recently developed CRISPR-based diagnostic assays. Cross-reactivity will be analyzed through comparison of target regions with the genome sequence of the human, seven coronaviruses and 21 other organisms. Inclusivity analysis will be performed through the verification of the sequence variability within the target regions using publicly available SARS-CoV-2 sequences from around the world. The absence of cross-reactivity and any mutations in target regions of the assay used would provide a higher degree of confidence in the CRISPR-based diagnostic tests being developed while the presence could help guide the assay development efforts. We believe that this study would provide potentially important information for clinicians, researchers, and decision-makers.

Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it is important to verify the genomic variability at primer/probe binding regions periodically. This step-by-step protocol describes a bioinformatics approach for an extensive evaluation of the sequence variability within the primer/probe target regions of the SARS-CoV-2 genome. The protocol can be applied to any molecular diagnostic assay of choice using freely available software programs and the ready-to-use multiple sequence alignment (MSA) file provided. Graphic abstract Overview of the sequence tracing protocol. The figure was created using the Library of Science and Medical Illustrations from somersault18:24 licensed under a CC BY-NC-SA 4.0 license (https://creativecommons.org/licenses/by-nc-sa/4.0/). Video abstract: https://youtu.be/M1lV1liWE9k.

TRK-Fused Gene (TFG), a protein involved in protein secretion pathways, is an essential component of the antiviral innate immune response.

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named as 2019-nCoV) is responsible for the recent COVID-19 pandemic and polymerase chain reaction (PCR) is the current standard method for its diagnosis from patient samples. This study conducted a reassessment of published diagnostic PCR assays, including those recommended by the World Health Organization (WHO), through the evaluation of mismatches with publicly available viral sequences. An exhaustive evaluation of the sequence variability within the primer/probe target regions of the viral genome was performed using more than 17 000 viral sequences from around the world. The analysis showed the presence of mutations/mismatches in primer/probe binding regions of 7 assays out of 27 assays studied. A comprehensive bioinformatics approach for in silico inclusivity evaluation of PCR diagnostic assays of SARS-CoV-2 was validated using freely available software programs that can be applied to any diagnostic assay of choice. These findings provide potentially important information for clinicians, laboratory professionals and policy-makers.