Presence of rare potential pathogenic variants in subjects under 60 years old with very severe or fatal COVID-19

Background/Objectives: Genetic variants affecting host defense against pathogens may help explain COVID-19 fatal outcomes. Our aim was to identify rare genetic variants related to COVID-19 severity in a selected group of patients under 60 years who required intubation or resulting in death. Method(s): Forty-four very severe COVID-19 patients were selected from the Spanish STOP-Coronavirus cohort, which comprises more than 3,500 COVID-19 patients. Genotype was performed by whole exome sequencing and variants were selected by using a gene panel of 867 candidate genes (immune response, primary immunodeficiencies or coagulation, among other). Variants were filtered, priorized and their potential pathogenicity was assessed following ACGM criteria. Result(s): We detected 44 different variants of interest, in 29 different patients (66%). Some of these variants were previously described as pathogenic (26%). Mostly, the candidate variants were located in genes related to immune response (38%), congenital disorders of glycosylation (14%) or damaged DNA binding genes (9%). A network analysis, showed three main components, consisting of 25 highly interconnected genes related to immune response and two additional networks enriched in carbohydrate metabolism and in DNA metabolism and repair processes. Conclusion(s): The variants identified affect different, but interrelated, functional pathways such as immune response and glycosylation. Further studies are needed for confirming the ultimate role of the new candidate genes described in the present study on COVID-19 severity.

The Principles of SARS-CoV-2 Intervariant Competition Are Exemplified in the Pre-Omicron Era of the Colombian Epidemic.

The first 18 months of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in Colombia were characterized by three epidemic waves. During the third wave, from March through August 2021, intervariant competition resulted in Mu replacing Alpha and Gamma. We employed Bayesian phylodynamic inference and epidemiological modeling to characterize the variants in the country during this period of competition. Phylogeographic analysis indicated that Mu did not emerge in Colombia but acquired increased fitness there through local transmission and diversification, contributing to its export to North America and Europe. Despite not having the highest transmissibility, Mu's genetic composition and ability to evade preexisting immunity facilitated its domination of the Colombian epidemic landscape. Our results support previous modeling studies demonstrating that both intrinsic factors (transmissibility and genetic diversity) and extrinsic factors (time of introduction and acquired immunity) influence the outcome of intervariant competition. This analysis will help set practical expectations about the inevitable emergences of new variants and their trajectories. IMPORTANCE Before the appearance of the Omicron variant in late 2021, numerous SARS-CoV-2 variants emerged, were established, and declined, often with different outcomes in different geographic areas. In this study, we considered the trajectory of the Mu variant, which only successfully dominated the epidemic landscape of a single country: Colombia. We demonstrate that Mu competed successfully there due to its early and opportune introduction time in late 2020, combined with its ability to evade immunity granted by prior infection or the first generation of vaccines. Mu likely did not effectively spread outside of Colombia because other immune-evading variants, such as Delta, had arrived in those locales and established themselves first. On the other hand, Mu's early spread within Colombia may have prevented the successful establishment of Delta there. Our analysis highlights the geographic heterogeneity of early SARS-CoV-2 variant spread and helps to reframe the expectations for the competition behaviors of future variants.

Covid-19 variants: Impact on transmissibility and virulence

The genetic evolution of SARS-CoV-2 began in February 2020, with G614 spike protein strains superseding D614 strains globally. Since then with each subsequent mutations, the SARS-CoV-2 variants of concern, namely Alpha, Beta, Gamma, Delta and Omicron, superseded the previous one to become the dominant strain during the pandemic. By the end of November 2022, the Omicron variant and its descendent lineages account for 99.9% of sequences reported globally. All five VOCs have mutations located in the RBD of the spike protein, resulting in increased affinity of the spike protein to the ACE2 receptors resulting in enhanced viral attachment and its subsequent entry into the host cells. In vitro studies showed the mutations in spike protein help increase the viral fitness, enhancing both transmissibility and replication. In general, Alpha, Beta, Gamma, and Delta variants, were reported with higher transmissibility of 43-90%, around 50%, 170-240%, or 130-170% than their co-circulating VOCs, respectively. The Omicron however was found to be 2.38 times and 3.20 times more transmissible than Delta among the fully-vaccinated and booster-vaccinated households. Even the SARS-Cov-2 Omicron subvariants appear to be inherently more transmissible than the ones before. With the broader distribution, enhanced evasion, and improved transmissibility, SARS-CoV-2 variants infection cause severe diseases due to immune escape from host immunity and faster replication. Reports have shown that each subsequent VOC, except Omicron, cause increased disease severity compared with those infected with other circulating variants. The Omicron variant infection however, appears to be largely associated with a lower risk of hospitalisation, ICU admission, mechanical ventilation, and even a shorter length of hospital stay. It has been shown that the relatively much slower replication of the Omicron variants in the lung, resulted in a less severe disease.Copyright © 2022, Malaysian Society of Pathologists. All rights reserved.

Whole-genome sequencing of SARS-CoV-2 isolates from symptomatic and asymptomatic individuals in Tanzania.

Background: Coronavirus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accounts for considerable morbidity and mortality globally. Paucity of SARS-CoV-2 genetic data from Tanzania challenges in-country tracking of the pandemic. We sequenced SARS-CoV-2 isolated in the country to determine circulating strains, mutations and phylogenies and finally enrich international genetic databases especially with sequences from Africa. Methods: This cross-sectional study utilized nasopharyngeal swabs of symptomatic and asymptomatic adults with positive polymerase chain reaction tests for COVID-19 from January to May 2021. Viral genomic libraries were prepared using ARTIC nCoV-2019 sequencing protocol version three. Whole-genome sequencing (WGS) was performed using Oxford Nanopore Technologies MinION device. In silico genomic data analysis was done on ARTIC pipeline version 1.2.1 using ARTIC nCoV-2019 bioinformatics protocol version 1.1.0. Results: Twenty-nine (42%) out of 69 samples qualified for sequencing based on gel electrophoretic band intensity of multiplex PCR amplicons. Out of 29 isolates, 26 were variants of concern [Beta (n = 22); and Delta (n = 4)]. Other variants included Eta (n = 2) and B.1.530 (n = 1). We found combination of mutations (S: D80A, S: D215G, S: K417N, ORF3a: Q57H, E: P71L) in all Beta variants and absent in other lineages. The B.1.530 lineage carried mutations with very low cumulative global prevalence, these were nsp13:M233I, nsp14:S434G, ORF3a:A99S, S: T22I and S: N164H. The B.1.530 lineage clustered phylogenetically with isolates first reported in south-east Kenya, suggesting regional evolution of SARS-CoV-2. Conclusion: We provide evidence of existence of Beta, Delta, Eta variants and a locally evolving lineage (B.1.530) from samples collected in early 2021 in Tanzania. This work provides a model for ongoing WGS surveillance that will be required to inform on emerging and circulating SARS-CoV-2 diversity in Tanzania and East Africa.

How concerning is a SARS-CoV-2 variant of concern? Computational predictions and the variants labeling system.

In this study, we evaluated the use of a predictive computational approach for SARS-CoV-2 genetic variations analysis in improving the current variant labeling system. First, we reviewed the basis of the system developed by the World Health Organization (WHO) for the labeling of SARS-CoV-2 genetic variants and the derivative adapted by the United States Centers for Disease Control and Prevention (CDC). Both labeling systems are based on the virus' major attributes. However, we found that the labeling criteria of the SARS-CoV-2 variants derived from these attributes are not accurately defined and are used differently by the two agencies. Consequently, discrepancies exist between the labels given by WHO and the CDC to the same variants. Our observations suggest that giving the variant of concern (VOC) label to a new variant is premature and might not be appropriate. Therefore, we used a comparative computational approach to predict the effects of the mutations on the virus structure and functions of five VOCs. By linking these data to the criteria used by WHO/CDC for variant labeling, we ascertained that a predictive computational comparative approach of the genetic variations is a good way for rapid and more accurate labeling of SARS-CoV-2 variants. We propose to label all emergent variants, variant under monitoring or variant being monitored (VUM/VBM), and to carry out computational predictive studies with thorough comparison to existing variants, upon which more appropriate and informative labels can be attributed. Furthermore, harmonization of the variant labeling system would be globally beneficial to communicate about and fight the COVID-19 pandemic.

Evolution of variants of concern of SARS-CoV-2 during three waves in Senegal

Introduction/ Background: In Senegal, SARS-CoV-2 incidence evolved with three successive epidemic waves. The first wave started on March 2020 with low virus variability whilst the second outbreak started in December was dominated by the Alpha variant. With the third taking place in June 2021, we investigated the involvement of other variants. Methods: During three waves of the pandemic, 163,788 nasopharyngeal swabs have been analysed at the Institut de Recherche en Santé, de Surveillance Epidémiologique et de Formations (IRESSEF). From those, 10,189 positive samples were screened with Seegene Real-time reverse-transcription polymerase chain reaction RT-PCR new variant. From the screened samples, 972 positives were sequenced and 10% of the negatives for detection of new variants. The ARTIC Network methodology with Oxford Nanopore Technologies (ONT) has been used for sequencing. Results: Our data have overall shown that the Senegalese strains are very similar to each other or closely related to other. During the first wave, the most common clade found was 19A (70.27%) and majority of the samples were of the B.1 (54.05) lineage. We noted more diversity during the second wave where clade 20B (40.82%) was more frequent, followed by clade 20A (28.91%), 20I (10.54%). At the level of lineages, we identified variants of interest as B.1.1.7 (10.54%), B.1.525 (6.12%), and B.1.617.2 (0.68%). In the third wave, we observed at the clade level with mainly 21D (47.69%) and 21A (20%). Impact: SARS-CoV-2 diversity may affect the virus's properties, such as it spreads, disease severity, performance of vaccines, tools, or other public health and social measures. Therefore, such tracking of SARS-CoV-2 variants highlight the role some African institutes like IRESSEF surveillance capabilities through real-time sequencing of SARS-CoV-2 genomes in the local context. Conclusion: In Senegal, the SARS-CoV-2 pandemic has disrupted the organization of the health system. IRESSEF contributed to put in place strategies to respond effectively to the expectations of medical authorities by providing them with data on the strains circulating in Senegal at each moment of the epidemic.

Systematic review of the epidemiological status and genomic analysis of SARS-CoV-2 isolated from domestic dogs and cats

The aim of the research was to carry out a systematic review about the epidemiology of SARS-CoV-2 infection in dogs and cats, as well as the genomic analysis of virus samples isolated from dogs and cats worldwide. For this, the systematic review was structured based on PRISMA's protocol. Articles were obtained using the following keywords: SARS-CoV-2, COVID-19, dogs, cats, epidemiology, animal transmission, pets, companion animals, animal reservoirs and zoonosis. Additionally, all of SARS-CoV-2 genomes isolated from dogs and cats worldwide, reported in GISAID's EpiCoV™ database, were selected and analyzed through Nextclade's tool for the generation of the respective phylogenetic trees. The exposure - natural infection with SARS-CoV-2 from January 2020 to October 2021 of 100 dogs and 108 cats positive by the RTq-PCR technique was reported worldwide. Furthermore, 141 SARS-CoV-2 genetic sequences have been isolated from dogs (50) and cats (91), where the following variants monitored by public health organizations were found: the variants of concern (VOC) Alpha, Gamma and Delta, and the variants of interest (VOI) Iota and Lambda. On the other hand, viral lineage B.1. has been predominantly isolated in both dogs and cats (13.3%) and North America is the region with the greatest number of SARS-CoV-2 genomes isolated from both species (43.6%). SARS-CoV-2 has the ability to infect domestic canines and felines, its exposure to VOCs: Alpha, Gamma and Delta, and VOIs: Iota and Lambda being of public health interest;probably due to a «spillover» effect from the human. However, these two species have a low capacity to transmit the virus to other susceptible species, considering that they can act as epidemiological dead-end hosts in the transmission dynamics of SARSCoV-2.

Bebtelovimab, Alone and Together with Bamlanivimab and Etesevimab, as a Broadly Neutralizing Monoclonal Antibody Treatment and a Slow Intravenous Push Option for Ambulatory COVID-19

RATIONALE: Bebtelovimab is a potent, fully human monoclonal antibody targeting the S-protein of SARS-CoV-2, with broad neutralizing activity to all variants of concern (including Omicron), based on non-clinical assays. This study aimed to evaluate the efficacy and safety of bebtelovimab alone (BEB) or together with bamlanivimab (BAM) and etesevimab (ETE) for the treatment of mild-tomoderate COVID-19, delivered via slow intravenous push.METHODS : This portion of the phase 2 BLAZE-4 trial (NCT4634409) enrolled 706 patients (between May and July 2021) with mild-tomoderate COVID-19 within 3 days of first laboratory diagnosis of SARS-CoV-2 infection. Patients at low-risk for severe COVID-19 were randomized 1:1:1 (double-blinded) to placebo, BEB 175 mg, or BEB 175 mg+BAM 700 mg+ETE 1400 mg (BEB+BAM+ETE). Patients at high-risk for severe COVID-19 were randomized 1:1 (open-label) to BEB or BEB+BAM+ETE;a subsequent treatment arm enrolled patients to BEB+BAM+ETE using CDC expanded criteria for high-risk. All treatments were administered intravenously over ≥30 seconds (open-label BEB) or ≥6.5 minutes (all other treatment arms). For the placebo-controlled population (termed low-risk), the primary endpoint was the proportion of patients with persistently high viral load (PHVL) (log viral load >5.27) on Day 7. For the open-label population (termed high-risk), the primary endpoint was safety outcomes and statistics were descriptive. RESULTS : Baseline sequencing data was available for 611 patients, 90.2% (n=551) aligned with a variant of interest or concern (WHO designation), with the majority infected with Delta (49.8%) and Alpha (28.6%) variants. For the low-risk population, active treatment arms had a numerically lower proportion of patients with PHVL compared to placebo, albeit not at a level of statistical signficance (see Table). Viral load-area under the curve analysis from baseline to Day 11 showed signficant reduction for patients treated with BEB compared to placebo. Time to sustained symptom resolution was significantly improved among patients who received BEB relative to placebo. As expected, the incidence of COVID-19-related hospitalization or all-cause deaths by day 29 were similar within the low-risk population. Overall, results were similar between patients in low-risk and high-risk populations receiving active treatment (see Table). The majority of treatment emergent adverse events (AEs) were mild-to-moderate in low-risk (n=36/380,9.5%) and high-risk patients (n=46/326,14.1%). Serious AEs were reported in 7/326 (2.1%) high-risk patients;none were reported in low-risk patients.CONCLUSION: The safety and efficacy data support the further development of bebtelovimab delivered via slow intravenous push of at least 30 seconds. (Table Presented).

Evolution of SARS-CoV-2 variants: a mini-review

SARS-CoV-2 has evolved over time with several mutations, especially on the spike protein, which has led to emergence of various variants. With the evolution of SARS-CoV-2 come new challenges in surveillance, effectiveness of preventive and treatment strategies, and outcome of the disease. Despite the lockdowns, mask mandates and other preventive measures put in place, in addition to over 10 million vaccine doses that have been administered globally as of February 2022, COVID-19 cases have risen to over 435 million and resulted in over 5.9 million deaths, largely as a result of the evolution of SARS-CoV-2 variants. To review the evolution of these variants, we searched different online database sources using keywords such as “source of SARS-CoV-2”, “SARS-CoV-2 origin”, “evolution of SARS-CoV-2”, “SARS-CoV-2 variants”, “variants of concern”, “variants of interest”, and “variants of high consequence”. This was to enable us give a good report about the various variants of SARS-CoV-2 that have emerged so far, and the public health challenges posed by them.

SARS-CoV-2 Circulation in Guinea, March 2020-July 2021

The SARS-CoV-2 genetic variants emergence doesn't spare the West African continent which has to face vaccination implementation delay. Beside classical qRT-PCR diagnostic testing, strengthening of sequencing capacity is the cornerstone for tracking and fighting the emergence of SARS-CoV-2 variants in real time. From March 12th, 2020 to July 16th, 2021, a panel of 136 full length genomes of SARS-CoV-2 mutants/variants present in human nasopharyngeal swab samples conserved in the Biobank of the Institut Pasteur De Guinée were sequenced using Illumina methodology. The Guinean sequences, originating from the general population, expatriates, and travelers, were distributed into 7 clades. During March- August 2020, the sequences were exclusively distributed into 2 clades, 20A and 20B, most originating from Europe. The 20D and 20C clades were furtively observed in October 2020 and February 2021 respectively. The SARS-CoV-2 variant of concern (VOC) 20I/B.1.1.7/Alpha was first identified in January 2021, increased in incidence up to March 2021, and then decreased from April to June 2021, corresponding to the dynamic described in Africa. The variant of interest (VOI) 21D/B.1.525/Eta originating from Nigeria circulated in February-May 2021. The 21A/B.1.617.2/Delta VOC was detected from May 2021 in Guinea, became dominant in July and persisted behind the present sampling over August and September 2021. A similar dynamic was globally observed in Africa resulting in a clear increase of lethality in the population. In contrast, other variants previously found in Africa, such as the 20H/B.1.351/Beta VOC and variants from the sublineage A (A.23.1 lineage from East Africa and the A.27 lineage), were not detected in this study. This overview of SARS-CoV-2 over 1.5 years in Guinea demonstrates that virus clades, VOC and VOI were progressively introduced, mostly by travelers through the Conakry Airport, before spreading through the country. The tracking of viral evolution by sequencing is a continuous task. Since November 2021, a new wave is related to the emergence of the VOC Omicron. Making countries autonomous in sequencing is a challenge in Africa, not only to fight Covid-19, but also to face the numerous other emerging zoonoses which circulate across the continent.

Exploratory Data Analysis on SARS-CoV-2 Variants in India: especially Omicron (B.1.1.529) as of 6th December 2021

SAR-CoV-2 is now spreading around the world, resulting in increased hospitalization and catastrophic fatality. The genome for coronavirus disease is vulnerable to abnormalities, which leads to genetic distortion and immunity loss. A novel variant of concern (VoC) with a new mutation having Pango lineage B.1.1.529, namely Omicron by WHO, was first found in South Africa at the end of November 2021. As of date, this new variant has already been spread rapidly in more than 58 countries and no doubt including India. In this work, Exploratory Data Analysis (EDA) analysis has been taken on different types of Covid-19 variants to date, where Omicron has demonstrated to be more increased transmissibility and infectious as compared to other variants. EDA offers several graphical representations to a better comprehension of the data and generates statistics for numerical data present in the dataset, as of 6th December 2021. Starting from 2nd December 2021 India has reported 23 new omicron cases within four days, which is a major challenge both for the doctors and government. Moreover, the EDA technique has been carried out for finding a significant correlation with the total number of Omicron cases as of the date in India using a scatter plot. Also, a conceptual design has been configured in this project that describes the whole process of how EDA analysis has been carried out and a Treemap that looks forward to outliers in all countries representing more than twenty-five covid-19 variants. © 2022 IEEE.

Mutational cascade of SARS-CoV-2 leading to evolution and emergence of omicron variant.

BACKGROUND: Emergence of new variant of SARS-CoV-2, namely omicron, has posed a global concern because of its high rate of transmissibility and mutations in its genome. Researchers worldwide are trying to understand the evolution and emergence of such variants to understand the mutational cascade events. METHODS: We have considered all omicron genomes (n = 302 genomes) available till 2nd December 2021 in the public repository of GISAID along with representatives of variants of concern (VOC), i.e., alpha, beta, gamma, delta, and omicron; variant of interest (VOI) mu and lambda; and variant under monitoring (VUM). Whole genome-based phylogeny and mutational analysis were performed to understand the evolution of SARS CoV-2 leading to emergence of omicron variant. RESULTS: Whole genome-based phylogeny depicted two phylogroups (PG-I and PG-II) forming variant specific clades except for gamma and VUM GH. Mutational analysis detected 18,261 mutations in the omicron variant, majority of which were non-synonymous mutations in spike (A67, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H), followed by RNA dependent RNA polymerase (rdrp) (A1892T, I189V, P314L, K38R, T492I, V57V), ORF6 (M19M) and nucleocapsid protein (RG203KR). CONCLUSION: Delta and omicron have evolutionary diverged into distinct phylogroups and do not share a common ancestry. While, omicron shares common ancestry with VOI lambda and its evolution is mainly derived by the non-synonymous mutations.

Molecular Epidemiology of SARS-CoV-2 in Tunisia (North Africa) through Several Successive Waves of COVID-19.

Documenting the circulation dynamics of SARS-CoV-2 variants in different regions of the world is crucial for monitoring virus transmission worldwide and contributing to global efforts towards combating the pandemic. Tunisia has experienced several waves of COVID-19 with a significant number of infections and deaths. The present study provides genetic information on the different lineages of SARS-CoV-2 that circulated in Tunisia over 17 months. Lineages were assigned for 1359 samples using whole-genome sequencing, partial S gene sequencing and variant-specific real-time RT-PCR tests. Forty-eight different lineages of SARS-CoV-2 were identified, including variants of concern (VOCs), variants of interest (VOIs) and variants under monitoring (VUMs), particularly Alpha, Beta, Delta, A.27, Zeta and Eta. The first wave, limited to imported and import-related cases, was characterized by a small number of positive samples and lineages. During the second wave, a large number of lineages were detected; the third wave was marked by the predominance of the Alpha VOC, and the fourth wave was characterized by the predominance of the Delta VOC. This study adds new genomic data to the global context of COVID-19, particularly from the North African region, and highlights the importance of the timely molecular characterization of circulating strains.

COVID-19 pandemic: the delta variant, T-cell responses, and the efficacy of developing vaccines.

BACKGROUND: The mayhem COVID-19 that was ushered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) was declared pandemic by the World Health Organization in March 2020. Since its initial outbreak in late 2019, the virus has affected hundreds of million adults in the world and killing millions in the process. After the approval of newly developed vaccines, severe challenges remain to manufacture and administer them to the adult population globally in quick time. However, we have witnessed several mutations of the virus leading to 'waves' of viral spread and mortality. WHO has categorized these mutations as variants of concern (VOCs) and variants of interest (VOIs). The mortality due to COVID-19 has also been associated with various comorbidities and improper immune response. This has created further complications in understanding the nature of the SARS-CoV2-host interaction that has fuelled doubts in the efficacy of the approved vaccines. Whether there is requirement of booster dose and whether the impending wave could affect the children are some of the hotly debated topics. MATERIALS AND METHODS: A systematic literature review of PubMed, Medline, Scopus, Google Scholar was utilized to understand the nature of Delta variant and how it alters our T-cell responses and cytokine production and neutralizes vaccine-generated antibodies. CONCLUSION: In this review, we discuss the variants of SARS-CoV2 with specific focus on the Delta variant. We also specifically review the T-cell response against the virus and bring a narrative of various factors that may hold the key to fight against this marauding virus.

Performance Characteristics of Sequencing Assays for Identification of the SARS-CoV-2 Viral Genome

Background. As the SARS-CoV-2 (SCV-2) virus evolves, diagnostics and vaccines against novel strains rely on viral genome sequencing. Researchers have gravitated towards the cost-effective and highly sensitive amplicon-based (e.g. ARTIC) and hybrid capture sequencing (e.g. SARS-CoV-2 NGS Assay) to selectively target the SCV-2 genome. We provide an in silico model to compare these 2 technologies and present data on the high scalability of the Research Use Only (RUO) workflow of the SARS-CoV-2 NGS Assay. Methods. In silico work included alignments of 383,656 high-quality genome sequences belonging to variant of concern (VOC) or variant of interest (VOI) isolates (GISAID). We profiled mismatches and sequencing dropouts using the ARTIC V3 primers, SARS-CoV-2 NGS Assay probes (Twist Bioscience) and 11 synthesized viral sequences containing mutations and compared the performance of these assays using clinical samples. Further, the miniaturized hybrid capture workflow was optimized and evaluated to support high-throughput (384-plex). The sequencing data was processed by COVID-DX software. Results. We detected 101,432 viruses (27%) with > = 1 mismatch in the last 6 base pairs of the 3' end of ARTIC primers;of these, 413 had > = 2 mismatches in one primer. In contrast, only 38 viruses (0.01%) had enough mutations ( > = 10) in a hybrid capture probe to have a similar effect on coverage. We observed that mutations in ARTIC primers led to complete dropout of the amplicon for 4/11 isolates and diminished coverage in additional 4. Twist probes showed uniform coverage throughout with little to no dropouts. Both assays detected a wide range of variants (~99.9% coverage at 5X depth) in clinical samples (CT value < 30) collected in NY (Spring 2020-Spring 2021). The distribution of the number of reads and on target rates were more uniform among specimens within amplicon-based sequencing. However, uneven genome coverage and primer dropouts, some in the spike protein, were observed on VOC/VOI and other isolates highlighting limitations of an amplicon-based approach. Conclusion. The RUO workflow of the SARS-CoV-2 NGS Assay is a comprehensive and scalable sequencing tool for variant profiling, yields more consistent coverage and smaller dropout rate compared to ARTIC (0.05% vs. 7.7%).

Characteristics of SARS-CoV-2 RNA Viral Loads among Nursing Home Residents and Staff with Repeat Positive Tests ≥ 90 Days after Initial Infection: 5 US Jurisdictions, July 2020-March 2021

Background. Background. Understanding the viral load and potential infectivity of individuals in nursing homes (NH) with repeat positive SARS-CoV-2 tests ≥ 90 days after initial infection has important implications for safety related to transmission in this high-risk setting. Methods. Methods. We collected epidemiologic data by reviewing records of a convenience sample of NH residents and staff with respiratory specimens who had positive SARS-CoV-2 rRT-PCR test results from July 2020 through March 2021 and had a SARS-CoV-2 infection diagnosed ≥ 90 days prior. No fully vaccinated individuals were included. Each contributed one repeat positive specimen ≥ 90 days after initial, which was sent to CDC and retested using rRT-PCR. Specimens were assessed for replication-competent virus in cell culture if Cycle threshold (Ct) < 34 and sequenced if Ct < 30. Using Ct values as a proxy for viral RNA load, specimens were categorized as high (Ct < 30) or low (if Ct ≥ 30 or rRT-PCR negative at retesting). Continuous variables were compared using Wilcoxon signed-rank tests. Proportions were compared using Chi-squared or Fisher's exact tests. Results. Results. Of 64 unvaccinated individuals with specimens from 61 unique NHs, 14 (22%) were sent for culture and sequencing. Ten of 64 (16%) had a high viral RNA load, of which four (6%) were culture positive and none were known variants of interest or concern (Figure 1). Median days to repeat positive test result were 122 (Interquartile range (IQR): 103-229) and 201 (IQR: 139-254), respectively, for high versus low viral load specimens (p=0.13). More individuals with high viral loads (5/10, 50%) reported COVID-19 symptoms than with a low viral load (1/27, 4%, p=0.003). Most individuals (46/58, 79%) were tested following known or suspected exposures, with no significant differences between high and low viral load (p=0.18). Conclusion. In this study, nearly 1 in 6 NH residents and staff with repeat positive tests after 90 days demonstrated high viral RNA loads and viable virus, indicating possible infectivity. While individuals with high RNA viral load may be more likely to be symptomatic, distinguishing asymptomatic individuals who have high viral loads may be difficult with timing since initial infection, other test results, or exposure history alone.

Identification of Complement-Related Missense Variants in Pediatric Patients with Acute and Post COVID-19 Syndromes

Background: Complement dysregulation has been documented in the molecular pathophysiology of COVID-19 and recently implicated in the relevant pediatric patient inflammatory responses. Aims: Based on our previous data in adults, we hypothesized that signatures of complement genetic variants would also be detectable in pediatric patients exhibiting COVID-19 signs and symptoms. Methods: We prospectively studied consecutive pediatric patients from our COVID-19 Units (November 2020-March 2021). COVID-19 was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Patient data were recorded by treating physicians that followed patients up to discharge. DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exons of complement-associated genes (C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MBL2, MASP1, MASP2, COLEC11, FCN1, FCN3 as well as ADAMTS13 and ΤHBD) spanning 15 bases into introns. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. A preliminary analysis (A) for the identification of variants of clinical significance was based on annotated ClinVar data, while a further and more selective analysis (B) was performed to identify missense complement coding variants that may biochemically contribute to the deregulation of innate responses during infection. This analysis was mainly based on the dbSNP and UniProt databases and available literature. Results: We studied 80 children and adolescents, 8 of whom developed inflammatory syndromes (MIS-C group). Among them, 41 were hospitalized and eventually all survived. 1. In our preliminary analysis, patients exhibited heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 97, range: 61-103). We found a variant of ADAMTS13 (rs2301612, missense) in 39 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (33 patients);and rs800292 in CFH (36 patients). Among them, 40 patients had a combination of these characterized variants. This combination was significantly associated with the presence of dyspnea (p=0.031) and cough (p=0.042). Furthermore, 27 patients had a pathogenic variant in MBL2 (rs1800450), and 7 a pathogenic deletion in FCN3 that have been previously associated with inflammatory syndromes. 2. The results of our further analysis are summarized in Figure. We identified common variants, some well represented by relatively high frequencies (>70%) (rs11098044 in CFI, rs1061170 in CFH and rs12711521 in MASP2) and others less abundant, but varying considerably between the hospitalized group, the non-admitted group and the MIS-C individuals (rs2230199 in C3, rs1065489 in CFH, rs12614 and rs641153 in CFB, rs1800450 in MBL2, rs2273346 and rs72550870 in MASP2, rs72549154 in MASP3 and rs7567833 in COLEC11, all highlighted in Figure in red).). Structurally, the majority of these common variants of interest encode charge reversal mutations. These may influence protein-protein interactions in complex formations that are important for complement activation and/or regulation. Conclusion: In pediatric COVID-19 we have detected a novel set of complement missense coding variants some of which have been implicated earlier in inflammatory syndromes and endothelial stress responses. Certain combinations of mutations of alternative and/or lectin pathway components may increase the threshold dynamics of complement consumption and therefore alter COVID-19 phenotypes. [Formula prese ted] Disclosures: Gavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy;Gilead Corporation: Honoraria;Pfizer Corporation: Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials;Sanofi: Other: clinical trials;Ocopeptides: Other: clinical trials;GSK: Other: clinical trials;Incyte: Other: clinical trials;Takeda: Other: clinical trials;Amgen: Other: clinical trials;Janssen: Other: clinical trials;novartis: Other: clinical trials;Celgene: Other: clinical trials;Roche: Other: clinical trials;Astellas: Other: clinical trials.

Epidemiological analysis of the emergence and disappearance of the SARS-CoV-2 Kappa variant within a region of British Columbia, Canada.

Background: The Kappa variant is designated as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of interest (VOI). We identified 195 Kappa variant cases in a region of British Columbia, Canada-the largest published cluster in North America. Objectives: To describe the epidemiology of the Kappa variant in relation to other circulating SARS-CoV-2 variants of concern (VOC) in the region to determine if the epidemiology of the Kappa variant supports a VOI or VOC status. Methods: Clinical specimens testing positive for SARS-CoV-2 collected between March 10 and May 2, 2021, were screened for the detection of known circulating VOCs; approximately 50% of specimens were subsequently selected for whole genome sequencing (WGS). Epidemiological analysis was performed comparing the characteristics of Kappa cases to the main circulating variants in the region (Alpha and Gamma) and to non-VOC/VOI cases. Results: A total of 2,079 coronavirus disease 2019 (COVID-19) cases were reported in the region during the study period, of which 54% were selected for WGS. The 1,131 sequenced cases were categorized into Kappa, Alpha, Gamma and non-VOC/VOI. While Alpha and Gamma cases were found to have a significantly higher attack rate among household contacts compared to non-VOI/VOC cases, Kappa was not. Conclusion: Epidemiological analysis supports the designation of Kappa as a VOI and not a VOC. The Alpha and Gamma variants were found to be more transmissible, explaining their subsequent dominance in the region and the rapid disappearance of the Kappa variant. Variant surveillance strategies should focus on both detection of established VOCs and detection of potential new VOCs.

SARS CoV-2;a closer look at the agent of the pandemic of modern times

Due to the COVID-19 pandemic, as of September 2021, a total of 222,309,456 people were infected in the world and a total of 4,592,685 patients were lost. The pandemic, which has a fatality rate of around 2%, has made and continues to make us live thhrough all experiences of epidemics that we have only read about in Annals of Medicine and Microbiology and that deeply affected the World at their times. The virus causing the pandemic has a positive polarity RNA genome of 30,000 bases and produces a total of 29 proteins. Of these proteins, 4 are structural, 16 are nonstructural, and 9 are accessory proteins. SARS-CoV-2 is an enveloped RNA virus with a diameter of 150-200 nm, has an S (spike-spike-tassel) glycoprotein on its surface, which, like other coronaviruses, creates the crown appearance unique to these viruses. After the S protein is synthesized as a polyprotein, it is cleaved into S1 and S2 subunits. The S1 subunit binds to the target cell, and the S2 subunit performs fusion with the cell membrane to be infected. Since these functions are critical features of a successful viral infection, the S protein is the main target of all interventions to prevent virus infection. In this context, the main target of neutralizing antibodies and drugs to stop virus infection before it starts is the S protein. The S protein has a trimer structure similar to hemagglutinin in influenza virus and contains the fusion peptide that becomes exposed during transition from the prefusion configuration to the fusion configuration and facilitates the fusion function with the cellular/endosomal membranes. Apart from the S protein, SARS-CoV-2 has structural proteins known as E (envelope), M (membrane), and N (nucleocapsid) proteins;The N protein binds to the RNA genome and together with the S, E and M proteins and the RNA genome form the virion. While SARS-CoV-2 S protein attaches to cells using Cellular Angiotensin Converting Enzyme 2 (HCoV- NL63, SARS-CoV and SARS-CoV-2), other coronaviruses use different receptors (Aminopeptidase N-HCoV-229E;dipeptidyl peptidase 4- MERS-CoV). Unlike viruses in this group, the SARS CoV-2 S1 protein with receptor binding domain (RBD) has a cleavage site made up of polybasic amino acids at the S1-S2 border and used by the cellular furin protease, which is believed to provide advantages to the virus in proteolytic cleavage, cell tropism, virulence and pathogenicity. ACE-2 is important in the renin-angiotensin-aldosterone system and although it is rarely found in the circulation, it is widely expressed in organs and is an enzyme involved in the regulation of blood pressure and fluid balance. Following intracellular entry and fusion of membranes, the SARS-CoV-2 genome is released into the cytoplasm and gene expression proceeds as a temporally and spatially well-regulated process. Non-structural proteins, which are produced from direct translation of ORF1a and ORF1b regions of positive sense genomic RNA, form the replication and transcription complex. These complexes establish the infrastructure for the next steps. The common features of coronaviruses such as cytoplasmic replication, viral gene expression through sub-genomic nested set messages, exocytosis of mature virions within vesicles occur in SARS-CoV-2 as well. One of the most important problems in the COVID-19 pandemic has been the emergence of variant viruses. These viruses adversely affecting the transmission rate, virulence, clinical course, and the effectiveness of the diagnostic or therapeutic methods carry mutations that lead to amino acid changes, especially in the RBD region. The World Health Organization and other authorities refer to these viruses as variants of concern or variants of interest. As of September 2021, WHO lists Alpha (UK, September 2020), Beta (South Africa, May 2020), Gamma (Brazil, November 2020), and Delta (India, October 2020) viruses as variants of concern. Also, Eta (December 2020), Iota (USA, November 2020), Kappa (India, October 2020), Lambda (Peru December, 2020) and Mu (Colombia, January 2021) mutant viruses are on he list variants of interest. In conclusion, less than 2 years of time has passed since the emergence of the COVID-19 agent SARS CoV-2 virus. However, this virus has been the most extensively studied viral agent in the history of medicine and the most detailed information has been gathered about the infection. Despite all these, it is difficult to indicate that the fight against this pathogen has been successful nor are we any closer to declare that the enormous danger the virus poses to humanity is reduced.

Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology.

The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the 'midnight' 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.