Strategic Retesting to Reduce False Positive SARS-CoV-2 PCR Test Results.

BACKGROUND: Nucleic acid amplification testing is the gold standard for SARS-CoV-2 diagnostics, although it may produce a certain number of false positive results. There has not been much published about the characteristics of false positive results. In this study, based on retesting, specimens that initially tested positive for SARS-CoV-2 were classified as true or false positive groups to characterize the distribution of cycle threshold (CT) values for N1 and N2 targets and number of targets detected for each group. METHODS: Specimens that were positive for N-gene on retesting and accompanied with S-gene were identified as true positives (true positive based on retesting, rTP), while specimens that retested negative were classified as false positives (false positive based on retesting, rFP). RESULTS: Of the specimens retested, 85/127 (66.9%) were rFP, 16/47 (34.0%) specimens with both N1 and N2 targets initially detected were rFP, and the CT values for each target was higher in rFP than in rTP. ROC curve analysis showed that optimal cutoff values of CT to differentiate between rTP and rFP were 34.8 for N1 and 33.0 for N2. With the optimal cutoff values of CT for each target, out of the 24 specimens that were positive for both N1 and N2 targets and classified as rTP, 23 (95.8%) were correctly identified as true positives. rFP specimens had a single N1 target in 52/61 (85.2%) and a single N2 target in 17/19 (89.5%). Notably, no true positive results were obtained from any specimens with only N2 target detected. CONCLUSIONS: These results suggest that retesting should be performed for positive results with a CT value greater than optimal cutoff value for each target or with a single N1 target amplified, considering the possibility of a false positive. This may provide guidance on indications to perform retesting to minimize the number of false positives.

LINC01002 functions as a ceRNA to regulate FRMD8 by sponging miR-4324 for the development of COVID-19.

BACKGROUND: Syndrome coronavirus-2 (SARS-CoV-2) has developed various strategies to evade the antiviral impact of type I IFN. Non-structural proteins and auxiliary proteins have been extensively researched on their role in immune escape. Nevertheless, the detailed mechanisms of structural protein-induced immune evasion have not been well elucidated. METHODS: Human alveolar basal epithelial carcinoma cell line (A549) was stimulated with polyinosinic-polycytidylic acid (PIC) and independently transfected with four structural proteins expression plasmids, including nucleocapsid (N), spike (S), membrane (M) and envelope (E) proteins. By RT-qPCR and ELISA, the structural protein with the most pronounced inhibitory effects on IFN-ß induction was screened. RNA-sequencing (RNA-Seq) and two differential analysis strategies were used to obtain differentially expressed genes associated with N protein inhibition of IFN-ß induction. Based on DIANA-LncBase and StarBase databases, the interactive competitive endogenous RNA (ceRNA) network for N protein-associated genes was constructed. By combining single-cell sequencing data (GSE158055), lncRNA-miRNA-mRNA axis was further determined. Finally, RT-qPCR was utilized to illustrate the regulatory functions among components of the ceRNA axis. RESULTS: SARS-CoV-2 N protein inhibited IFN-ß induction in human alveolar epithelial cells most significantly compared with other structural proteins. RNA-Seq data analysis revealed genes related to N protein inhibiting IFNs induction. The obtained 858 differentially expressed genes formed the reliable ceRNA network. The function of LINC01002-miR-4324-FRMD8 axis in the IFN-dominated immune evasion was further demonstrated through integrating single-cell sequencing data. Moreover, we validated that N protein could reverse the effect of PIC on LINC01002, FRMD8 and miR-4324 expression, and subsequently on IFN-ß expression level. And LINC01002 could regulate the production of FRMD8 by inhibiting miR-4324. CONCLUSION: SARS-CoV-2 N protein suppressed the induction of IFN-ß by regulating LINC01002 which was as a ceRNA, sponging miR-4324 and participating in the regulation of FRMD8 mRNA. Our discovery provides new insights into early intervention therapy and drug development on SARS-CoV-2 infection.

Development of Detection Antibody Targeting the Linear Epitope in SARS-CoV-2 Nucleocapsid Protein with Ultra-High Sensitivity.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.

Near-infrared responsive gold nanorods for highly sensitive colorimetric and photothermal lateral flow immuno-detection of SARS-CoV-2.

The highly infectious characteristics of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlight the necessity of sensitive and rapid nucleocapsid (N) protein-based antigen testing for early triage and epidemic management. In this study, a colorimetric and photothermal dual-mode lateral flow immunoassay (LFIA) platform for the rapid and sensitive detection of the SARS-CoV-2 N protein was developed based on gold nanorods (GNRs), which possessed tunable local surface plasma resonance (LSPR) absorption peaks from UV-visible to near-infrared (NIR). The LSPR peak was adjusted to match the NIR emission laser 808 nm by controlling the length-to-diameter ratio, which could maximize the photothermal conversion efficiency and achieve photothermal detection signal amplification. Qualitative detection of SARS-CoV-2 N protein was achieved by observing the strip color, and the limit of detection was 2 ng mL-1, while that for photothermal detection was 0.096 ng mL-1. Artificial saliva samples spiked with the N protein were analyzed with the recoveries ranging from 84.38% to 107.72%. The intra-assay and inter-assay coefficients of variation were 6.76% and 10.39%, respectively. We further evaluated the reliability of this platform by detecting 40 clinical samples collected from nasal swabs, and the results matched well with that of nucleic acid detection (87.5%). This method shows great promise in early disease diagnosis and screening.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy.

Lateral flow immunoassays (LFIAs) are an essential and widely used point-of-care test for medical diagnoses. However, commercial LFIAs still have low sensitivity and specificity. Therefore, we developed an automatic ultrasensitive dual-color enhanced LFIA (DCE-LFIA) by applying an enzyme-induced tyramide signal amplification method to a double-antibody sandwich LFIA for antigen detection. The DCE-LFIA first specifically captured horseradish peroxidase (HRP)-labeled colored microspheres at the Test line, and then deposited a large amount of tyramide-modified signals under the catalytic action of HRP to achieve the color superposition. A limit of detection (LOD) of 3.9 pg/mL and a naked-eye cut-off limit of 7.8 pg/mL were achieved for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein. Additionally, in the inactivated virus detections, LOD equivalent to chemiluminescence (0.018 TCID50/mL) was obtained, and it had excellent specificity under the interference of other respiratory viruses. High sensitivity has also been achieved for detection of influenza A, influenza B, cardiac troponin I, and human chorionic gonadotrophin using this DCE-LFIA, suggesting the assay is universally applicable. To ensure the convenience and stability in practical applications, we created an automatic device. It provides a new practical option for point-of-care test immunoassays, especially ultra trace detection and at-home testing.

Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

ConspectusThe COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

Characterizing a visual lateral flow device for rapid SARS-CoV-2 virus protein detection: pre-clinical and system assessment.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections have affected more than 769 million individuals worldwide over the last few years. Although the pandemic is transitioning into an endemic, the COVID-19 outbreak is still a global concern. A rapid screening platform is needed for effective preventive and control measures. Herein, a visual rapid lateral flow platform for SARS-CoV-2 nucleocapsid protein detection is developed. Under optimal conditions, the system demonstrated good detection sensitivity and selectivity against tested respiratory viruses. The system provides direct visual detection with a limit of 0.7 ng of the nucleocapsid protein per mL of a sample (0.7 ng mL-1) within 15 minutes. Further, a correlation between direct visual detection and semi-quantitative analysis using a reader showed a similar detection limit (R2 = 0.9571). The repeatability and reproducibility studies highlighted the potential of the system for the rapid screening of SARS-CoV-2 infection, with variations within 5% and 10% at high and low protein concentrations, respectively. Subsequent pre-clinical validation to correlate the performance with the standard molecular approach (RT-PCR) using 170 nasopharyngeal swabs demonstrated 98% estimated sensitivity (95% CI, 89.35-99.95%) and 100% specificity (95% CI, 96.38-100%). The positive and negative predictive values were reported to be 100% and 99%, respectively, with an accuracy of 99.3%. With high viral load samples (Ct value ≤25, n = 47), the system demonstrated 100% detection sensitivity and specificity. The proposed technique provides a valuable platform for potential use in rapid screening, particularly during pandemics, where diagnostic capacity and mass screening are crucial.

A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody.

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

TRIM6 facilitates SARS-CoV-2 proliferation by catalyzing the K29-typed ubiquitination of NP to enhance the ability to bind viral genomes.

The Nucleocapsid Protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not only the core structural protein required for viral packaging, but also participates in the regulation of viral replication, and its post-translational modifications such as phosphorylation have been shown to be an important strategy for regulating virus proliferation. Our previous work identified NP could be ubiquitinated, as confirmed by two independent studies. But the function of NP ubiquitination is currently unknown. In this study, we first pinpointed TRIM6 as the E3 ubiquitin ligase responsible for NP ubiquitination, binding to NP's CTD via its RING and B-box-CCD domains. TRIM6 promotes the K29-typed polyubiquitination of NP at K102, K347, and K361 residues, increasing its binding to viral genomic RNA. Consistently, functional experiments such as the use of the reverse genetic tool trVLP model and gene knockout of TRIM6 further confirmed that blocking the ubiquitination of NP by TRIM6 significantly inhibited the proliferation of SARS-CoV-2. Notably, the NP of coronavirus is relatively conserved, and the NP of SARS-CoV can also be ubiquitinated by TRIM6, indicating that NP could be a broad-spectrum anti-coronavirus target. These findings shed light on the intricate interaction between SARS-CoV-2 and the host, potentially opening new opportunities for COVID-19 therapeutic development.

Intrinsic factors behind long COVID: IV. Hypothetical roles of the SARS-CoV-2 nucleocapsid protein and its liquid-liquid phase separation.

When the SARS-CoV-2 virus infects humans, it leads to a condition called COVID-19 that has a wide spectrum of clinical manifestations, from no symptoms to acute respiratory distress syndrome. The virus initiates damage by attaching to the ACE-2 protein on the surface of endothelial cells that line the blood vessels and using these cells as hosts for replication. Reactive oxygen species levels are increased during viral replication, which leads to oxidative stress. About three-fifths (~60%) of the people who get infected with the virus eradicate it from their body after 28 days and recover their normal activity. However, a large fraction (~40%) of the people who are infected with the virus suffer from various symptoms (anosmia and/or ageusia, fatigue, cough, myalgia, cognitive impairment, insomnia, dyspnea, and tachycardia) beyond 12 weeks and are diagnosed with a syndrome called long COVID. Long-term clinical studies in a group of people who contracted SARS-CoV-2 have been contrasted with a noninfected matched group of people. A subset of infected people can be distinguished by a set of cytokine markers to have persistent, low-grade inflammation and often self-report two or more bothersome symptoms. No medication can alleviate their symptoms efficiently. Coronavirus nucleocapsid proteins have been investigated extensively as potential drug targets due to their key roles in virus replication, among which is their ability to bind their respective genomic RNAs for incorporation into emerging virions. This review highlights basic studies of the nucleocapsid protein and its ability to undergo liquid-liquid phase separation. We hypothesize that this ability of the nucleocapsid protein for phase separation may contribute to long COVID. This hypothesis unlocks new investigation angles and could potentially open novel avenues for a better understanding of long COVID and treating this condition.

Nucleocapsid and Spike Protein-Based Anti-SARS-CoV-2 Assay Performance in the Minority and Rural Coronavirus Insights Study: Characteristics of Socioeconomically Disadvantaged Populations with Health Disparities.

BACKGROUND: COVID-19 has had a devastating impact on Black, Hispanic, and other underserved, disadvantaged populations. Here anti-SARS-CoV-2 tests are characterized in disadvantaged patients to examine equivalence in US populations. METHODS: Underserved participant adults (age > 18 years) were enrolled before the availability of SARS-CoV-2 vaccines in Federal Qualified Health Centers in California, Florida, Louisiana, Illinois, and Ohio and contributed samples to the Minority and Rural Coronavirus Insights Study (MRCIS). A subset coined the MRCIS SARS-CoV-2 Antibody Cohort of 2365 participants was tested with the Roche Anti-SARS-CoV-2 assay (Cobas e601). Five hundred ninety-five of these were also tested with the Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG assay (VITROS-5600); 1770 were also tested with the Abbott ARCHITECT SARS-CoV-2 IgG assay (ARCHITECT-2000). Assay-specific cutoffs classified negative/positive results. RESULTS: Eight point four percent (199/2365) of the MRCIS SARS-CoV-2 Antibody Cohort was SARS-CoV-2 RNA positive at enrollment. Agreement between the Ortho/Roche and the Abbott/Roche antibody testing did not vary by enrollment RNA status. The Ortho (anti-spike protein) vs Roche (anti-nucleocapsid protein) comparison agreed substantially: kappa = 0.63 (95% CI: 0.57-0.69); overall agreement, 83%. However, agreement was even better for the Abbott vs Roche assays (both anti-nucleocapsid protein tests): kappa = 0.85 (95% CI: 0.81-0.87); overall agreement, 95%. Anti-SARS-CoV-2 comparisons stratified by demographic criteria demonstrated no significant variability in agreement by sex, race/ethnicity, or age. CONCLUSIONS: Analytical agreement is 96.4% for anti-spike-protein vs anti-nucleocapsid-protein comparisons. Physiologically, seroreversion of anti-nucleocapsid reactivity after infection occurred in the disadvantaged population similarly to general populations. No anti-SARS-CoV-2 assays included demonstrated a clinically significant difference due to the demographics of the disadvantaged MRCIS SARS-CoV-2 Antibody Cohort.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

The disordered N-terminal tail of SARS-CoV-2 Nucleocapsid protein forms a dynamic complex with RNA.

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.

PGAM5 degrades PDCoV N protein and activates type I interferon to antagonize viral replication.

IMPORTANCE: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

Prevalence and genetic variation of the M, N, and S2 genes of feline coronavirus in Shandong Province, China.

Feline coronavirus (FCoV) is the causative agent of feline infectious peritonitis and diarrhoea in kittens worldwide. In this study, a total of 73 feline diarrhoeal faecal samples were collected from animal hospitals and pet markets in ShanDong province from 2017 to 2019. FCoV was detected in 58.23% (46/73) of the samples, using the RT-PCR method. The results showed that the detection rate of FCoV in healthy cats and sick cats was 41.7% (10/24) and 81.6% (40/49), respectively. Full gene amplification and sequencing of the N, M, and S2 genes of FCoV isolates were performed. An amino acid mutation (M1058L) in the S2 gene was found that can be used as a marker for distinguishing feline enteric coronavirus (FECV) from feline infectious peritonitis virus (FIPV). This study provides new epidemiological information about FCoV that will aid in the prevention of FCoV in China.

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

TRIM21 promotes ubiquitination of SARS-CoV-2 nucleocapsid protein to regulate innate immunity.

The innate immune response is the first line of host defense against viral infections, but its role in immunity against SARS-CoV-2 remains unclear. By using immunoprecipitation coupled with mass spectroscopy, we observed that the E3 ubiquitin ligase TRIM21 interacted with the SARS-CoV-2 nucleocapsid (N) protein and ubiquitinated it at Lys375 . Upon determining the topology of the TRIM21-mediated polyubiquitination chain on N protein, we then found that polyubiquitination led to tagging of the N protein for degradation by the host cell proteasome. Furthermore, TRIM21 also ubiquitinated the N proteins of SARS-CoV-2 variants of concern, including Alpha, Beta, Gamma, Delta, and Omicron together with SARS-CoV and MERS-CoV variants. Herein, we propose that ubiquitylation and degradation of the SARS-CoV-2 N protein inhibited SARS-CoV-2 viral particle assembly, by which it probably involved in preventing cytokine storm. Eventually, our study has fully revealed the association between the host innate immune system and SARS-CoV-2 N protein, which may aid in developing novel SARS-CoV-2 treatment strategies.