RESUMEN
In this report, we describe the development of an RT-qPCR assay, termed Alpha Delta assay, which can detect SARS-COV-2 (SC-2) 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 N gene, and the Delta reaction targets the spike gene 156-158 mutations. Additionally, we developed a second Delta-specific assay, used as a confirmatory test for the Alpha Delta assay that targets the 119-120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15-25 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, we show that the Alpha Delta and Orf8-119del assays correctly identified the presence of Alpha and Delta lineages RNA in wastewater samples. 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.
RESUMEN
Emerging SARS-CoV-2 variants may threaten global vaccination efforts and awaited reduction in outbreak burden. In this study, we report a novel variant carrying the L452R mutation that emerged from a local B.1.362 lineage, B.1.362+L452R. The L452R mutation is associated with the Delta and Epsilon variants and was shown to cause increased infection and reduction in neutralization in pseudoviruses. Indeed, the B.1.362+L452R variant demonstrated a X4-fold reduction in neutralization capacity of sera from BNT162b2-vaccinated individuals compared to a wild-type strain. The variant infected 270 individuals in Israel between December 2020 and March 2021, until diminishing due to the gain in dominance of the Alpha variant in February 2021. This study demonstrates an independent, local emergence of a variant carrying a critical mutation, L452R, which may have the potential of becoming a variant of concern and emphasizes the importance of routine surveillance and detection of novel variants among efforts undertaken to prevent further disease spread.
RESUMEN
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 activities and provide valuable data for epidemiological and policy decision making. We developed a multiplex quantitative RT-qPCR (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, and one that detects the 242-244 deletion, which is specific to variant B.1.351. The fourth reaction identifies 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 assays specificity was tested against a panel of respiratory pathogens (n=16), showing high specificity towards SC-2 RNA. The assays 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.
RESUMEN
Routine detection, surveillance and reporting of SARS-CoV-2 novel variants is important, as these threaten to hinder vaccination efforts. Herein we report a local novel strain that includes a non-synonymous mutation in the spike (S) protein - P681H and additional synonymous mutations. The P681H Israeli strain has not been associated with higher infection rates and was neutralized by sera from vaccinated individuals in comparable levels to the B.1.1.7 strain and a non-P681H strain from Israel.