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Background: Amidst the persistent global health threat posed by the evolving SARS-CoV-2 virus throughout the four-year-long COVID-19 pandemic, the focus has now turned to the Omicron variant and its subvariant, JN.1, which has rapidly disseminated worldwide. This study reports on the characteristics and clinical manifestations of patients during the surge of the JN.1 variant in Saudi Arabia; it also investigates the evolution of SARS-CoV-2 variants in organ transplant patients and identifies patient risk factors., Methods: A total of 151 nasopharyngeal samples from patients with PCR-confirmed SARS-CoV-2 infection were collected between September 2023 and January 2024. Demographic and clinical data of the patients were obtained from electronic health records. All confirmed positive samples underwent sequencing using Ion GeneStudio and the Ion AmpliSeq™ SARS-CoV-2 panel., Results: During the surge of the JN.1 variant, the average age of the patients was 40 years, ranging from 3 to 93 years, and nearly 50% of the patients were male. Our investigation revealed that the J.N variant predominantly infected patients with comorbidities or organ transplant recipients (57.6%). Moreover, patients with comorbidities or organ transplants exhibited a higher number of mutations. In our organ transplant cohort, an increased total number of spike mutations was associated with a lower risk of developing severe disease (OR = 0.96, 95% CI: 0.93-0.98)., Conclusions: Although JN.1 may not prove to be particularly harmful, it is crucial to recognize the persistent emergence of concerning variants, which create new pathways for the virus to evolve. The ongoing evolution of SARS-CoV-2 is evident in the continuous divergence of these variants from the original strain that marked the onset of the pandemic nearly four years ago., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: To describe the chronological genomic evolution of SARS-CoV-2 and its impact on public health in the Middle East and North Africa (MENA) region., Methods: This study analysed all available SARS-CoV-2 genomic sequences, metadata and rates of COVID-19 infection from the MENA region retrieved from the Global Initiative on Sharing All Influenza Data database from January 2020 to August 2021. Inferential and ‎descriptive statistics were conducted to describe the epidemiology of SARS-CoV-2., Results: Genomic surveillance of SARS-CoV-2 in the MENA region indicated that the variants in January 2020 predominately belonged to the G, GR, GH or O clades and that the most common variant of concern was Alpha. By August 2021, however, the GK clade dominated (57.4% of all sequenced genomes), followed by the G clade (18.7%) and the GR clade (11.6%). In August, the most commonly sequenced variants of concern were Delta in the Middle East region (91%); Alpha (44.3%) followed by Delta (29.7%) and Beta (25.3%) in the North Africa region; and Alpha (88.9%), followed by Delta (10%) in the fragile and conflict-affected regions of MENA. The mean proportion of the variants of concern among the total sequenced samples differed significantly by country (F=1.93, P=0.0112) but not by major MENA region (F=0.14, P=0.27) or by vaccination coverage (F=1.84, P=0.176)., Conclusion: This analysis of the genomic surveillance of SARS-CoV-2 provides an essential description the virus evolution and its impact on public health safety in the MENA region. As of August 2021, the Delta variant showed a genomic advantage in the MENA region. The MENA region includes several fragile and conflict-affected countries with extremely low levels of vaccination coverage and little genomic surveillance, which may soon exacerbate the existing health crisis within those countries and globally., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Coronaviridae is a large family of enveloped, positive-strand RNA viruses that has plagued the world since it was discovered in humans in the 1960s. The recent severe acute respiratory syndrome coronavirus (SARS-CoV)-2 pandemic has already exceeded the number of combined cases and deaths witnessed during previous SARS-CoV and Middle East respiratory syndrome-CoV epidemics in the last two decades. This narrative review focuses on genomic mutations in SARS-CoV-2 and their impact on the severity and progression of COVID-19 in light of reported data in the literature. Notable SARS-CoV-2 mutations associated with open reading frames, the S glycoprotein, and nucleocapsid protein, currently circulating globally, are discussed along with emerging mutations such as those in the SARS-CoV-2 VUI 202012/01 variant in the UK and other European countries, the 484K.V2 and P.1 variants in Brazil, the B.1.617 variant in India, and South African variants 501Y.V2 and B.1.1.529 (omicron). These variants have the potential to influence the receptor binding domain, host-virus fusion, and SARS-CoV-2 replication. Correlating these mutations with disease dynamics could help us understand their pathogenicity and design appropriate therapeutics.

Introduction: In December 2019, a new severe acute respiratory syndrome coronavirus, SARS-CoV-2, emerged in China, causing coronavirus disease 2019. The present study investigated genetic profiles and variations of SARS-CoV-2 distributed in different regions of Saudi Arabia to begin to understand the pathogenesis and transmission of SARS-CoV-2 in this country and analyzed associations of these variations with host factors., Methodology: In total, 774 SARS-CoV-2 genomic sequences obtained and annotated by the Global Initiative on Sharing All Influenza Data (GISAID) were captured and analyzed., Results: The most common SARS-CoV-2 clades in Saudi Arabia were GH followed by O, GR, G, and S. Statistically significant associations were detected between clades and patient outcome. Age, as a host factor, was significantly associated with many variables, including virus geographical location, clade, and patient outcome. The most common variants detected were the NSP12_P323L mutation 94.9%, followed by the D614G mutation (76%) and the NS3_Q57H mutation (71.4%). The concerned variants B.1.1.7, B.1.351, and P.1 were not detected in our population. D614G was associated with higher morbidities than the wild-type virus, including higher rates of death and hospitalization. The NS3_Q57H mutation was the only variant associated with better patient outcome than the wild type. Risk of death was highest with the NSP12_P323L mutation (OR = 1.84; 95% CI = 0.37-9.30) and lowest with the NS3_Q57H mutation (OR = 0.43; 95% CI = 0.25-0.727)., Conclusions: SARS-CoV-2 has evolved uniquely and independently in Saudi Arabia. Our findings provide evidence to begin linking the evolutionary implications to host factors and their effects on the virus severity and transmission., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Dalia Abdullah Obeid, Madain Saleh Alsanea, Rawan Talal Alnemari, Ahmed Ali Al-Qahtani, Sahar Isa Althawadi, Maysoon Saleh Mutabagani, Reem Saad Almaghrabi, Faten Mohammed Alhadheq, Basma Mohammed Alahideb, Fatimah Saeed Alhamlan.)

Background: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol., Methods: We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory., Results: The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R 2 = 0.89)., Conclusions: These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pantazi, Al-Qahtani, Alhamlan, Alothaid, Matou-Nasri, Sourvinos, Vergadi and Tsatsanis.)

The genome of severe acute respiratory coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has undergone a rapid evolution, resulting in the emergence of multiple SARS-CoV-2 variants with amino acid changes. This study aimed to sequence the whole genome of SARS-CoV-2 and detect the variants present in specimens from Saudi Arabia. Furthermore, we sought to analyze and characterize the amino acid changes in the various proteins of the identified SARS-CoV-2 variants. A total of 1161 samples from patients diagnosed with COVID-19 in Saudi Arabia, between 1 April 2021 and 31 July 2023, were analyzed. Whole genome sequencing was employed for variant identification and mutation analysis. The statistical analysis was performed using the Statistical Analytical Software SAS, version 9.4, and GraphPad, version 9.0. This study identified twenty-three variants and subvariants of SARS-CoV-2 within the population, with the Omicron BA.1 (21K) variant (37.0%) and the Delta (21J) variant (12%) being the most frequently detected. Notably, the Omicron subvariants exhibited a higher mean mutation rate. Amino acid mutations were observed in twelve proteins. Among these, the spike (S), ORF1a, nucleocapsid (N), and ORF1b proteins showed a higher frequency of amino acid mutations compared to other the viral proteins. The S protein exhibited the highest incidence of amino acid mutations (47.6%). Conversely, the ORF3a, ORF8, ORF7a, ORF6, and ORF7b proteins appeared more conserved, demonstrating the lowest percentage and frequency of amino acid mutations. The investigation of structural protein regions revealed the N-terminal S1 subunit of the S protein to frequently harbor mutations, while the N-terminal domain of the envelope (E) protein displayed the lowest mutation frequency. This study provides insights into the variants and genetic diversity of SARS-CoV-2, underscoring the need for further research to comprehend its genome evolution and the occurrence of mutations. These findings are pertinent to the development of testing approaches, therapeutics, and vaccine strategies. [ABSTRACT FROM AUTHOR]

RNA viruses, including SARS-CoV-2, rely on genetic mutation as a major evolutionary mechanism, leading to the emergence of variants. Organ transplant recipients (OTRs) may be particularly vulnerable to such mutations, making it crucial to monitor the spread and evolution of SARS-CoV-2 in this population. This cohort study investigated the molecular epidemiology of SARS-CoV-2 by comparing the SARS-CoV-2 whole genome, demographic characteristics, clinical conditions, and outcomes of COVID-19 illness among OTRs (n = 19) and non-OTRs with (n = 38) or without (n = 30) comorbid conditions. Most patients without comorbidities were female, whereas most OTRs were male. Age varied significantly among the three groups: patients with comorbidities were the oldest, and patients without comorbidities were the youngest. Whole-genome sequencing revealed that OTRs with mild disease had higher numbers of unusual mutations than patients in the other two groups. Additionally, OTRs who died had similar spike monoclonal antibody resistance mutations and 3CLpro mutations, which may confer resistance to nirmatrelvir, ensitrelvir, and GC37 therapy. The presence of those unusual mutations may impact the severity of COVID-19 illness in OTRs by affecting the virus's ability to evade the immune system or respond to treatment. The higher mutation rate in OTRs may also increase the risk of the emergence of new virus variants. These findings highlight the importance of monitoring the genetic makeup of SARS-CoV-2 in all immunocompromised populations and patients with comorbidity. [ABSTRACT FROM AUTHOR]

Introduction: In this study, coagulation and fibrinolysis parameters and their association with disease severity were investigated in coronavirus disease (COVID-19) patients. Materials and Methods: COVID-19 patients (n = 446) admitted to our institute between 21 February 2021 and 17 March 2022, were recruited. Clinical data and staging were collected from all patients. Blood samples were collected and analyzed for several parameters of fibrinolysis and coagulation, including alpha-2-antiplasmin(α2AP) and plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), D-dimer, and fibrinogen levels. Results: The TAFI, fibrinogen, and tPA levels were significantly higher in participants who died compared to that of patients who recovered (p < 0.001). However, PAI-1, tPA, and TAFI were significantly higher in patients admitted to the ICU than those of the healthy controls (p < 0.001 for PAI-1 and tPA; p = 0.0331 for TAFI). Our results showed that stage C and D COVID-19 patients had significantly higher levels of PAI-1 (p = 0.003). Furthermore, stage D COVID-19 patients had significantly higher tPA and TAFI values (p = 0.003). Conclusions: Hypofibrinolysis was the most prevalent condition among patients with severe COVID-19. In this study, several coagulation markers were elevated, making them suitable prognostic markers for hypofibrinolysis. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 genomic mutations outside the spike protein that may increase transmissibility and disease severity have not been well characterized. This study identified mutations in the nucleocapsid protein and their possible association with patient characteristics. We analyzed 695 samples from patients with confirmed COVID-19 in Saudi Arabia between 1 April 2021, and 30 April 2022. Nucleocapsid protein mutations were identified through whole genome sequencing. 2 tests and t tests assessed associations between mutations and patient characteristics. Logistic regression estimated the risk of intensive care unit (ICU) admission or death. Of the 60 mutations identified, R203K was the most common, followed by G204R, P13L, E31del, R32del, and S33del. These mutations were associated with reduced risk of ICU admission. P13L, E31del, R32del, and S33del were also associated with reduced risk of death. By contrast, D63G, R203M, and D377Y were associated with increased risk of ICU admission. Most mutations were detected in the SR-rich region, which was associated with low risk of death. The C-tail and central linker regions were associated with increased risk of ICU admission, whereas the N-arm region was associated with reduced ICU admission risk. Consequently, mutations in the N protein must be observed, as they may exacerbate viral infection and disease severity. Additional research is needed to validate the mutations' associations with clinical outcomes. [ABSTRACT FROM AUTHOR]

In late December 2019, the world woke to a reality of a pandemic of Coronavirus Disease (COVID-19), elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which belongs to a group of β-coronavirus. The potential to cause life-threatening respiratory failure and rapid transmission puts COVID-19 in the list of Public Health Emergency of International Concern (PHEIC). In the last two decades, this is the 3rd deadliest Coronavirus pandemic, following SARS which lasted between 2002 and 2003 and Middle East Respiratory Syndrome (MERS) from 2012 till date. Globally and as of April 23rd 2020, COVID-19 has affected 2,544,792 individuals in over 200 countries, causing 175,694 fatalities. While the SARS-CoV-2 originated in China with 84,302 confirmed cases and 4642 deaths as at the time of writing this review, the rapid transmission of SARS-CoV-2 has resulted in exponential increase in the number of cases outside of China to about 10 times the report case and death in mainland China. SARS-CoV-2 is suspected to be zoonotic in nature as genetic studies have shown sequence similarity to viruses originating from bats. Extreme precautionary measures, such as curfew, shutting of borders and quarantining of individuals suspected to be infected have been instituted with immediate effect; however, due to individuals that are asymptomatic, uncontrolled human-to-human transmission has resulted in exponential infection rate and numerous loss of lives even with this lockdown measures. This review article summarizes the developing situation surrounding the SARS-CoV-2 pandemic with respect to its epidemiology, unique genomic structure, possible origins, transmission, pathogenesis, comparison with other deadly species of Coronaviruses (CoV) and emerging treatment strategies built on informed literature. [ABSTRACT FROM AUTHOR]

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), from Wuhan, China, in December 2019 has challenged many countries. The current pandemic caused by this coronavirus has already negatively affected millions of people and the economies of countries worldwide. However, the challenges faced by Saudi Arabia during the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemic that began in 2012 led to marked improvements in the government's response to the current pandemic. Saudi Arabia is one of largest countries in the Middle East and is home to the holiest Muslim sites. Since the global risk of the virus was declared by the World Health Organization (WHO), Saudi Arabia has taken substantial public health measures to control the spread of the infection. This review reports on the transmission of SARS-COV-2 in Saudi Arabia and the proactive responses taken by the government, comparing the Saudi government's actions and their effects with those of other countries. Although Saudi Arabia is currently experiencing the peak of the pandemic, their early precautionary responses have shortened the period of individual/family isolation, reduced the number of confirmed infections and infection-related fatality rates, and decreased the economic burden of the people and the country compared with other countries in the Middle East and elsewhere. [ABSTRACT FROM AUTHOR]