everse-transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of respiratory samples is the gold standard for coronavirus disease 2019 (COVID-19) diagnosis 1 , but it has limitations. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 RNA) levels in the upper respiratory tract rapidly decrease after infection 2 while lower respiratory tract levels remain high 3 . RT-qPCR assays performed after early SARS-CoV-2 infection may thus yield false negatives, but infection events are often unclear, complicating interpretation. Nasopharyngeal tissue highly expresses angiotensin-converting enzyme 2 (ref. 4 ), the primary receptor for SARS-CoV-2 (ref. 5 ), but this protein is also expressed in other tissues (for example, cardiac and small intestine 6 ) reported to develop SARS-CoV-2 infections and related pathology 7 . Nasopharyngeal RT-qPCR assay results thus may not accurately reflect the status of lower respiratory tract or extrapulmonary infections.Circulating SARS-CoV-2 RNA detectable in mild to severe COVID-19 correlates with and predicts disease severity 8-10 , and appears responsible for extrapulmonary infections 7 . However, RT-qPCR exhibits poor overall sensitivity for SARS-CoV-2 RNA in plasma (≤41%) 11 . More sensitive and robust blood-based SARS-CoV-2 RNA assays compatible with routine clinical tests could thus improve COVID-19 diagnosis and prognostic evaluation. However, only two studies employing droplet digital RT-PCR, which is not suited for clinical use, have used ultrasensitive approaches to detect SARS-CoV-2 RNA in serum or plasma.Infected cells can secrete extracellular vesicles (EVs) containing pathogen-derived factors 12,13 , protecting these factors from hydrolases and allowing them to accumulate in the circulation 14 . Hepatitis

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Background: Circulating SARS-CoV-2 RNA may represent a more reliable indicator of infection than nasal RNA, but RT-qPCR lacks diagnostic sensitivity for blood samples. Methods: A CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHP) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for COVID-19 diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons. Results: CRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days post-infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n=159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, while RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified COVID-19 patients with one or more negative nasal swab RT-qPCR result. Conclusion: Results of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.

SARS-CoV-2 variants of concern (VOCs) that increase transmission or disease severity or reduce diagnostic or vaccine efficacy, continue to emerge across the world. Current methods available to rapidly detect these can be resource-intensive and thus sub-optimal for large-scale deployment needed during a pandemic response. Here, we describe a CRISPR-based assay that detects mutations in spike gene CRISPR PAM motif or seed regions to identify a pan-specific VOC single nucleotide polymorphism (SNP; D614G) and Alpha- and Delta-specific (S982A and D950N) SNPs. This assay exhibits good diagnostic sensitivity and strain specificity with nasal swabs and is designed for use in laboratory and point-of-care settings. This should enable rapid, high-throughput VOC identification required for surveillance and characterization efforts to inform clinical and public health decisions. Further, the assay can be adapted to target similar SNPs associated with emerging SARS-CoV-2 VOCs, or other rapidly evolving viruses.

Hepatitis C virus (HCV) infection compromises the natural defense mechanisms of the liver leading to a progressive end stage disease such as cirrhosis and hepatocellular carcinoma (HCC). The hepatic stress response generated due to viral replication in the endoplasmic reticulum (ER) undergoes a stepwise transition from adaptive to pro-survival signaling to improve host cell survival and liver disease progression. The minute details of hepatic pro-survival unfolded protein response (UPR) signaling that contribute to HCC development in cirrhosis are unknown. This study shows that the UPR sensor, the protein kinase RNA-like ER kinase (PERK), mediates the pro-survival signaling through nuclear factor erythroid 2-related factor 2 (NRF2)-mediated signal transducer and activator of transcription 3 (STAT3) activation in a persistent HCV infection model of Huh-7.5 liver cells. The NRF2-mediated STAT3 activation in persistently infected HCV cell culture model resulted in the decreased expression of hepatocyte nuclear factor 4 alpha (HNF4A), a major liver-specific transcription factor. The stress-induced inhibition of HNF4A expression resulted in a significant reduction of liver-specific microRNA-122 (miR-122) transcription. It was found that the reversal of hepatic adaptive pro-survival signaling and restoration of miR-122 level was more efficient by interferon (IFN)-based antiviral treatment than direct-acting antivirals (DAAs). To test whether miR-122 levels could be utilized as a biomarker of hepatic adaptive stress response in HCV infection, serum miR-122 level was measured among healthy controls, and chronic HCV patients with or without cirrhosis. Our data show that serum miR-122 expression level remained undetectable in most of the patients with cirrhosis (stage IV fibrosis), suggesting that the pro-survival UPR signaling increases the risk of HCC through STAT3-mediated suppression of miR-122. In conclusion, our data indicate that hepatic pro-survival UPR signaling suppresses the liver-specific HNF4A and its downstream target miR-122 in cirrhosis. These results provide an explanation as to why cirrhosis is a risk factor for the development of HCC in chronic HCV infection.