Sanger Sequencing for Molecular Diagnosis of SARS-CoV-2 Omicron Subvariants and Its Challenges

Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The European Centre for Disease Prevention and Control (eCDC) and the World Health Organization (WHO) recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the polymerase chain reaction (PCR)-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490 - 509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariants when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.2 RBD and an atypical BA.1 NTD, which can only be detected by using specially designed PCR primers. In addition, Sanger sequencing may reveal unclassified subvariants, such as BA.4/BA.5 with L84I mutation in the S gene NTD. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.

A spike-trimer protein-based tetravalent COVID-19 vaccine elicits enhanced breadth of neutralization against SARS-CoV-2 Omicron subvariants and other variants

Multivalent vaccines combining crucial mutations from phylogenetically divergent variants could be an effective approach to defend against existing and future SARS-Co V-2 variants.In this study,we developed a tetravalent COVID-19 vaccine SCTV01E,based on the trimeric Spike protein of SARS-Co V-2 variants Alpha,Beta,Delta,and Omicron BA.1,with a squalenebased oil-in-water adjuvant SCT-VA02B.In the immunogenicity studies in na?ve BALB/c and C57BL/6J mice,SCTV01E exhibited the most favorable immunogenic characteristics to induce balanced and broad-spectrum neutralizing potencies against pre-Omicron variants(D614G,Alpha,Beta,and Delta)and newly emerging Omicron subvariants(BA.1,BA.1.1,BA.2,BA.3,and BA.4/5).Booster studies in C57BL/6J mice previously immunized with D614G monovalent vaccine demonstrated superior neutralizing capacities of SCTV01E against Omicron subvariants,compared with the D614G booster regimen.Furthermore,SCTV01E vaccination elicited na?ve and central memory T cell responses to SARS-Co V-2 ancestral strain and Omicron spike peptides.Together,our comprehensive immunogenicity evaluation results indicate that SCTV01E could become an important COVID-19 vaccine platform to combat surging infections caused by the highly immune evasive BA.4/5 variants.SCTV01E is currently being studied in a head-to-head immunogenicity comparison phase 3 clinical study with inactivated and m RNA vaccines(NCT05323461).

Global challenge with the SARS-CoV-2 omicron BA.2(B.1.1.529.2)subvariant:Should we be concerned?

BA.2 is a novel omicron offshoot of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)that has gone viral.There is limited knowledge regarding this variant of concern.Current evidence suggests that this variant is more contagious but less severe than previous SARS-CoV-2 variants.However,there is concern regarding the virus mutations that could influence pathogenicity,transmissibility,and immune evasion.

Quantification and Differentiation of SARS-CoV‑2 Variants in Wastewater for Surveillance

Wastewater surveillance plays an important role in the monitoring of infections of SARS-CoV-2 at the community level.We report here the determination of SARS-CoV-2 and differentiation of its variants of concern in 294 wastewater samples collected from two major Canadian cities from May 2021 to March 2023.The overall method of analysis involved extraction of the virus and viral components using electronegative membranes,in situ stabilization and concentration of the viral RNA onto magnetic beads,and direct analysis of the viral RNA on the magnetic beads.Multiplex reverse transcription quantitative polymerase chain reaction(RT-qPCR)assays,targeting specific and naturally selected mutations in SARS-CoV-2,enabled detection and differentiation of the Alpha,Beta,Gamma,Delta,and Omicron variants.An Omicron triplex RT-qPCR assay targeting three mutations,HV 69−70 deletion,K417N,and L452R,was able to detect and differentiate the Omicron BA.1/BA.3,BA.2/XBB,and BA.4/5.This assay had efficiencies of 90−104%for all three mutation targets and a limit of detection of 28 RNA copies per reaction.Analyses of 294 wastewater samples collected over a two-year span showed the concentrations and trends of Alpha,Beta,Gamma,Delta,and Omicron variants as they emerge in two major Canadian cities participating in the wastewater surveillance program.The trends of specific variants were consistent with clinical reports for the same period.At the beginning of each wave,the corresponding variants were detectable in wastewater.For example,RNA concentrations of the BA.2 variant were as high as 10^(4)copies per 100 mL of wastewater collected in January 2022,when approximately only 50−60 clinical cases of BA.2 infection were reported in Canada.These results show that the strategy and highly sensitive assays for the variants of concern in wastewater are potentially useful for the detection of newly emerging SARS-CoV-2 variants and other viruses for future community biomonitoring.

An imported human case with the SARS-CoV-2 Omicron subvariant BA.2.75 in Yunnan Province,China

The Omicron variants spread rapidly worldwide after being initially detected in South Africa in November 2021.It showed increased transmissibility and immune evasion with far more amino acid mutations in the spike(S)protein than the previously circulating variants of concern(VOCs).Notably,on 15 July 2022,we monitored the first VOC/Omicron subvariant BA.2.75 in China from an imported case.Moreover,nowadays,this subvariant still is predominant in India.It has nine additional mutations in the S protein compared to BA.2,three of which(W152R,G446S,and R493Q reversion)might contribute to higher transmissibility and immune escape.This subvariant could cause wider spread and pose a threat to the global situation.Our timely reporting and continuous genomic analysis are essential to fully elucidate the characteristics of the subvariant BA.2.75 in the future.