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The emergence of the SARS-CoV-2 variant BA.2.86.1 raised a considerable concern, due to the large number of potentially virulent mutations. In this study, we developed a novel assay that specifically detects variant BA.2.86.1, and used it to screen environmental samples from wastewater treatment sites across Israel. By using a multiplex assay that included a general SARS-CoV-2 reaction, together with the BA.2.86.1-specific reaction and a control reaction, we quantified the absolute number of viral copies in each sample and its relative abundance, compared with the total copy number of circulating SARS-CoV-2. Evaluation of the new reactions showed that they are both sensitive and specific, detecting down to four copies per reaction, and maintain specificity in the presence of Omicron variants BA.1, 2 and 4 RNA. Examination of 279 samples from 30 wastewater collection sites during August-September 2023 showed that 35 samples (12.5 %) were positive, from 18 sites. Quantitative analysis of the samples showed that the relative abundance of variant BA.2.86.1 with respect to the total viral load of SARS-CoV-2 was very low and consisted between 0.01 % and 0.6 % of the total SARS-CoV-2 circulation. This study demonstrates the importance of combining wastewater surveillance with the development of specialized diagnostic assays, when clinical testing is insufficient. This approach may be useful for timely response by public health authorities in future outbreaks., 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 Elsevier B.V. All rights reserved.)

The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants. One leading hypothesis suggests these variants may have emerged during chronic infections of immunocompromised individuals, but limited data from these cases hinders comprehensive analyses. Here, we harnessed millions of SARS-CoV-2 genomes to identify potential chronic infections and used language models (LM) to infer chronic-associated mutations. First, we mined the SARS-CoV-2 phylogeny and identified chronic-like clades with identical metadata (location, age, and sex) spanning over 21 days, suggesting a prolonged infection. We inferred 271 chronic-like clades, which exhibited characteristics similar to confirmed chronic infections. Chronic-associated mutations were often high-fitness immune-evasive mutations located in the spike receptor-binding domain (RBD), yet a minority were unique to chronic infections and absent in global settings. The probability of observing high-fitness RBD mutations was 10-20 times higher in chronic infections than in global transmission chains. The majority of RBD mutations in BA.1/BA.2 chronic-like clades bore predictive value, i.e., went on to display global success. Finally, we used our LM to infer hundreds of additional chronic-like clades in the absence of metadata. Our approach allows mining extensive sequencing data and providing insights into future evolutionary patterns of SARS-CoV-2., (© 2024. The Author(s).)

Although recombination is a feature of coronavirus evolution, previously detected recombinant lineages of SARS-CoV-2 have shown limited circulation thus far. Here, we present a detailed phylogenetic analysis of four SARS-CoV-2 lineages to investigate the possibility of virus recombination among them. Our analyses reveal well-supported phylogenetic differences between the Orf1ab region encoding viral non-structural proteins and the rest of the genome, including Spike (S) protein and remaining reading frames. By accounting for several deletions in NSP6, Orf3a, and S, we conclude that the B.1.628 major cluster, now designated as lineage XB, originated from a recombination event between viruses of B.1.631 and B.1.634 lineages. This scenario is supported by the spatiotemporal distribution of these lineages across the USA and Mexico during 2021, suggesting that the recombination event originated in this geographical region. This event raises important questions regarding the role and potential effects of recombination on SARS-CoV-2 evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: Despite the success in developing COVID-19 vaccines, containment of the disease is obstructed worldwide by vaccine production bottlenecks, logistics hurdles, vaccine refusal, transmission through unvaccinated children, and the appearance of new viral variants. This underscores the need for effective strategies for identifying carriers/patients, which was the main aim of this study., Methods: We present a bubble-based PCR testing approach using swab-pooling into lysis buffer. A bubble is a cluster of people who can be periodically tested for SARS-CoV-2 by swab-pooling. A positive test of a pool mandates quarantining each of its members, who are then individually tested while in isolation to identify the carrier(s) for further epidemiological contact tracing., Results: We tested an overall sample of 25 831 individuals, divided into 1273 bubbles, with an average size of 20.3 ± 7.7 swabs/test tube, obtaining for all pools (≤37 swabs/pool) a specificity of 97.5% (lower bound 96.6%) and a sensitivity of 86.3% (lower bound 78.2%) and a post hoc analyzed sensitivity of 94.6% (lower bound 86.7%) and a specificity of 97.2% (lower bound 96.2%) in pools with ≤25 swabs, relative to individual testing., Discussion: This approach offers a significant scale-up in sampling and testing throughput and savings in testing cost, without reducing sensitivity or affecting the standard PCR testing laboratory routine. It can be used in school classes, airplanes, hospitals, military units, and workplaces, and may be applicable to future pandemics., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this report, we describe the development of a reverse transcription-quantitative PCR (RT-qPCR) assay, termed Alpha-Delta assay, which can detect all severe acute respiratory syndrome coronavirus 2 (SC-2) variants and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in the N gene, and the Delta reaction targets the spike gene 156 to 158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha-Delta assay that targets the 119 to 120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15 to 25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha-Delta assay and the Orf8 119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole-genome sequencing. Lastly, the capability of the Alpha-Delta assay and Orf8 119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive, and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health. IMPORTANCE The new assays described herein enable rapid, straightforward, and cost-effective detection of severe acute respiratory syndrome coronavirus 2 (SC-2) with immediate classification of the examined sample as Alpha, Delta, non-Alpha, or non-Delta variant. This is highly important for two main reasons: (i) it provides the scientific and medical community with a novel diagnostic tool to rapidly detect and classify any SC-2 sample of interest as Alpha, Delta, or none and can be applied to both clinical and environmental samples, and (ii) it demonstrates how to respond to the emergence of new variants of concern by developing a variant-specific assay. Such assays should improve our preparedness and adjust the diagnostic capacity to serve clinical, epidemiological, and research needs.

Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens ( n  = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.