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The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVβ3 and α5β1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVβ3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.

Inass Taha,1 Yasser Abdou,2 Ikhlas Hammad,2 Omnia Nady,2 Gamal Hassan,2 Magdy F Farid,3 Fadwa S Alofi,4 Najla Alharbi,2 Emad Salamah,5 Nawaf Aldeeb,2 Ghaidaa Elmehallawy,1 Rehab Alruwathi,6 Elmaghraby Sarah,7 Alhusainin Rashad,2 Ola Rammah,2 Hassan Shoaib,2 Mohammed ElSagheer Omar,2 Yara Elmehallawy,8 Saba Kassim9 1Department of Medicine, Faculty of Medicine, Taibah University, Al-Madinah Al-Munawara, Saudi Arabia; 2Department of Medicine, Ohud Hospital, Al-Madinah Al-Munawara, Saudi Arabia; 3ICU, Ohud Hospital, Al-Madinah Al-Munawara, Saudi Arabia; 4Department of Medicine, Infectious Disease Unit, King Fahad Hospital, Al-Madinah Al-Munawara, Saudi Arabia; 5Department of Urology, Ohud Hospital, Al-Madinah Al-Munawara, Saudi Arabia; 6Department of OB&GYN, MMCH Hospital, King Salman Medical City, Al-Madinah Al-Munawara, Saudi Arabia; 7Department of Medicine, Prince Mohammad Bin Abdulaziz National Guard Hospital, Al-Madinah Al-Munawara, Saudi Arabia; 8Department of Medicine, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia; 9Department of Preventive Dental Sciences, College of Dentistry, Taibah University, Al-Madinah Al-Munawara, Saudi ArabiaCorrespondence: Inass Taha, Email itaha@taibahu.edu.saBackground: Most patients admitted to intensive care units (ICUs) with severe Corona Virus Disease 2019 (COVID-19) pneumonia receive antibacterial antibiotics with little evidence of bacterial infections.Objective: This study was designed to review the profiles of patients with severe COVID-19 pneumonia requiring intensive care, the rate of bacterial coinfection, the antibiotics used, and their relation to patient outcomes (death or recovery).Methods: This was a retrospective study that reviewed the medical records of all patients with confirmed COVID-19 (n = 120) severe pneumonia admitted directly from the emergency room to the intensive care unit, at a public hospital during the period from May 2020 to April 2021. The data collected included patients’ demographic and laboratory data, comorbidities, antibiotic treatment, and their outcome. Descriptive statistics, bivariate inferential analysis tests (chi-square and unpaired T-Tests) and multivariable binary logistic regression were performed.Results: The mean age of the patients was 56.8 ± 16.5 years old, and among them, 74 (62.7%) were males. Of the included patients, 92 (77.0%) had comorbidities, 76 (63.3%) required mechanical ventilation and 30 (25%) died. All patients received empirical antibiotics for suspected bacterial coinfection. The most common antibiotics used were azithromycin (n = 97, 8%) and imipenem (n = 83, 9%). Ninety patients (75%) were on two empirical antibiotics. Early positive cultures for pathogens were found only in four patients (3.3%), whereas 36 (30%) patients had positive cultures 5– 10 days after admission. The most frequently isolated pathogens were Acinetobacter baumannii (n = 16) and coagulase-negative Staphylococci (n = 14). In bivariate analysis empirical treatment with azithromycin resulted in a significantly lower mortality rate (p = 0.023), meanwhile mechanical ventilation, days of stay in intensive care unit, morbidities (e.g., lung disease), linezolid and, vancomycin use associated with mortality (p< 0.05). The adjusted logistic regression, controlling for age and gender, revealed that azithromycin antibiotic was more likely protective from mortality (OR= 0.22, 95%CI 0.06-0.85, p=0.028. However, patients with lung diseases and under mechanical ventilation were 35.21 and 19.57 more likely to die (95%CI =2.84-436.70, p=0.006; 95%CI=2.66-143.85, p=0.003, respectively).Conclusion: Bacterial coinfection with severe COVID-19 pneumonia requiring intensive care was unlikely. The benefit of Azithromycin over other antibiotics could be attributed to its anti-inflammatory properties rather than its antibacterial effect.Keywords: coronavirus disease 2019, antibiotics, intensive care unit, bacterial coinfection

SummaryMonitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. The availability of patient hospital records is crucial for linking the genomic sequence information to virus function during the course of infections. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. From the assembled sequences, we estimate the SARS-CoV-2 effective population size and infection rate and outline the epidemiological dynamics of import and transmission events during this period in Saudi Arabia. We show that two consecutive mutations (R203K/G204R) in the SARS-CoV-2 nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein by mass-spectrometry analysis. Furthermore, analysis of the host cell transcriptome suggests that the mutant N protein results in dysregulated interferon response genes. We provide crucial information in linking the R203K/G204R mutations in the N protein as a major modulator of host-virus interactions and increased viral load and underline the potential of the nucleocapsid protein as a drug target during infection.

Molecular diagnosis and surveillance of pathogens such as SARS‐CoV‐2 depend on nucleic acid isolation. Pandemics at the scale of COVID‐19 can cause a global shortage of proprietary commercial reagents and BSL‐2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open‐source method, magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real‐world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol‐chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID‐19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS‐CoV‐2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field‐deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens., One important bottleneck in the diagnosis and surveillance of COVID‐19 is the shortage of kits for RNA extraction. Magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS) is an open‐source, safe, fast, and scalable method for RNA extraction. MAVRICS rivals commercial kits but requires minimal materials, and thus could become an enabling technology for widespread community testing of diverse pathogens.

The Coronavirus Disease 2019 (COVID-19), caused by the novel SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Immunological surrogate markers, in particular antigen-specific responses, are of unquestionable value for clinical management of patients with COVID-19. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized patients with RT-PCR confirmed COVID-19 infection. Our data show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Notably, anti-S and -N IgG, peaked 20-40 day after disease onset, and were still detectable for at least up to 70 days, with nAbs observed during the same time period. Moreover, nAbs titers were strongly correlated with IgG antibodies. Significantly higher levels of nAbs as well as anti-S1 and N IgG and IgM antibodies were found in patients with more severe clinical presentations, patients requiring admission to intensive care units (ICU) or those with fatal outcomes. Interestingly, lower levels of antibodies, particularly anti-N IgG and IgM in the first 15 days after symptoms onset, were found in survivors and those with mild clinical presentations. Collectively, these findings provide new insights into the characteristics and kinetics of antibody responses in COVID-19 patients with different disease severity.

Background: The Coronavirus Disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread globally. Although several commercial SARS-CoV-2 rapid serological assays have been developed, little is known about their performance and accuracy in detecting SARS-CoV-2 specific antibodies in COVID-19 patient samples. Method: We have evaluated the performance of seven commercially available rapid lateral flow immunoassay (LFIA) serological assays obtained from different manufacturers, and compared them to in-house developed and validated ELISA assays for the detection of SARS-CoV-2 specific IgG and IgM antibodies in COVID-19 patients. Results: While all evaluated LFIA assays showed high specificity, our data showed a significant variation in sensitivity of these assays in which it ranged from 0 to 54% for samples collected early during infection (3-7 days post symptoms onset) and from 54 to 88% for samples collected at later time points during infection (8-27 days post symptoms onset). Conclusion: Commercially available LFIA assays for detection of SARS-CoV-2 specific antibodies may be specific and show high degree of variation in their sensitivity. Further evaluations and validation of rapid serological assays is needed before being routinely used in detecting IgM and IgG in COVID-19 patients.

Highlights • RT-LAMP coupled with CRISPR-Cas12 provides a sensitive and specific virus detection platform. • iSCAN sensitivity and specificity are comparable with RT-qPCR. • iSCAN is a 1 h detection module that can help in testing in low resource areas. • iSCAN can be developed as a one-pot assay. • iSCAN reagents can be produced locally and deployed for SARS-CoV2 detection., The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript™ III Platinum™ One-step RT-qPCR and TaqPath™ 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility.

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method calledMagnetic-nanoparticle-AidedViralRNAIsolation ofContagiousSamples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. MAVRICS is scalable and thus could become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics.

As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

Background Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly-specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. Methods We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed NIRVANA. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus, and monitor mutations for up to 96 samples in real-time. Findings NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per μl of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2 positive samples mirror the epidemiology of COVID-19. Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and PMMoV (an omnipresent virus and water quality indicator) in municipal wastewater samples. Conclusions NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. Funding M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01, M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436)., Graphical Abstract, Bi et al. develop a convenient sequencing-based method that can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor viral mutations for up to 96 samples in real-time. This rapid and field-deployable method promises to help contain the spread of COVID-19 and ensure the effectiveness of vaccines.

As the coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus SARS-CoV-2, continues to spread rapidly around the world, there is an urgent need for validated serological assays to evaluate viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological tests to study the antibody response in COVID-19 patients. In order to validate the assays, we determined the cut-off values, sensitivity and specificity of the developed assays using sera collected from COVID-19 patients in Saudi Arabia at different time points after disease onset, as well as sera that are seropositive to other human CoVs; namely MERS-CoV, hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1. The SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N) ELISAs that we developed here not only showed high specificity and sensitivity, but also did not show any cross-reactivity with other CoVs. We also showed that all RT-PCR confirmed COVID-19 patients included in our study developed both virus specific IgM and IgG as early as one week after the onset of disease. The availability of these validated assays will enable us to determine the nature and duration of the antibody response mounted in response to SARS-CoV-2 infection. It will also allow conducting large-scale epidemiological studies to determine evidence of previous exposure to the virus and assess the true extent of virus spread within communities.

Background: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly-specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. Methods: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed NIRVANA. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus, and monitor mutations for up to 96 samples in real-time. Findings: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per µl of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2 positive samples mirror the epidemiology of COVID-19. Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and PMMoV (an omnipresent virus and water quality indicator) in municipal wastewater samples. Conclusions: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. Funding: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01, M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436).

A critical bottleneck in COVID‐19 diagnosis and surveillance is the shortage of kits for RNA extraction. In article number 2000068, Mo Li and co‐workers develop a DIY, open‐source, safe, and fast method for magnetic‐nanoparticle‐aided viral RNA isolation from contagious samples (MAVRICS). MAVRICS rivals commercial kits in performance and could become an enabling technology for community testing and wastewater monitoring in the current and future pandemics.