Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger.

INTRODUCTION: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. METHODS: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. RESULTS: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. CONCLUSION: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection.

The emergence of SARS-CoV-2 variants stresses the continued need for broad-spectrum therapeutic antibodies. Several therapeutic monoclonal antibodies or cocktails have been introduced for clinical use. However, unremitting emerging SARS-CoV-2 variants showed reduced neutralizing efficacy by vaccine induced polyclonal antibodies or therapeutic monoclonal antibodies. In our study, polyclonal antibodies and F(ab')2 fragments with strong affinity produced after equine immunization with RBD proteins produced strong affinity. Notably, specific equine IgG and F(ab')2 have broad and high neutralizing activity against parental virus, all SARS-CoV-2 variants of concern (VOCs), including B.1.1,7, B.1.351, B.1.617.2, P.1, B.1.1.529 and BA.2, and all variants of interest (VOIs) including B.1.429, P.2, B.1.525, P.3, B.1.526, B.1.617.1, C.37 and B.1.621. Although some variants weaken the neutralizing ability of equine IgG and F(ab')2 fragments, they still exhibited superior neutralization ability against mutants compared to some reported monoclonal antibodies. Furthermore, we tested the pre-exposure and post-exposure protective efficacy of the equine immunoglobulin IgG and F(ab')2 fragments in lethal mouse and susceptible golden hamster models. Equine immunoglobulin IgG and F(ab')2 fragments effectively neutralized SARS-CoV-2 in vitro, fully protected BALB/c mice from the lethal challenge, and reduced golden hamster's lung pathological change. Therefore, equine pAbs are an adequate, broad coverage, affordable and scalable potential clinical immunotherapy for COVID-19, particularly for SARS-CoV-2 VOCs or VOIs.

Uptake of severe acute respiratory syndrome coronavirus 2 spike protein mediated by angiotensin converting enzyme 2 and ganglioside in human cerebrovascular cells.

Introduction: There is clinical evidence of neurological manifestations in coronavirus disease-19 (COVID-19). However, it is unclear whether differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/spike protein (SP) uptake by cells of the cerebrovasculature contribute to significant viral uptake to cause these symptoms. Methods: Since the initial step in viral invasion is binding/uptake, we used fluorescently labeled wild type and mutant SARS-CoV-2/SP to study this process. Three cerebrovascular cell types were used (endothelial cells, pericytes, and vascular smooth muscle cells), in vitro. Results: There was differential SARS-CoV-2/SP uptake by these cell types. Endothelial cells had the least uptake, which may limit SARS-CoV-2 uptake into brain from blood. Uptake was time and concentration dependent, and mediated by angiotensin converting enzyme 2 receptor (ACE2), and ganglioside (mono-sialotetrahexasylganglioside, GM1) that is predominantly expressed in the central nervous system and the cerebrovasculature. SARS-CoV-2/SPs with mutation sites, N501Y, E484K, and D614G, as seen in variants of interest, were also differentially taken up by these cell types. There was greater uptake compared to that of the wild type SARS-CoV-2/SP, but neutralization with anti-ACE2 or anti-GM1 antibodies was less effective. Conclusion: The data suggested that in addition to ACE2, gangliosides are also an important entry point of SARS-CoV-2/SP into these cells. Since SARS-CoV-2/SP binding/uptake is the initial step in the viral penetration into cells, a longer exposure and higher titer are required for significant uptake into the normal brain. Gangliosides, including GM1, could be an additional potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

Transmisión del SARS-CoV-2 en escolares durante el primer trimestre del curso 2021/2022 en la provincia de Albacete: incidencia e impacto de las nuevas variantes.

OBJECTIVE: The objective of this study was to know the evolution of the COVID-19 pandemic in the school setting, and the effect of the new variants on it. DESIGN: It is an observational longitudinal descriptive study during the first term of the academic year 21/22. SITE: Community health services. PARTICIPANTS: Preschool, elementary, secondary, and high school students. INTERVENTIONS: none. MAIN MEASUREMENTS: We calculated cumulative incidence stratified by grade, source of infection and follow-up period, percentage of vaccinated cases and correlation between cumulative incidence in schooled children per week and cumulative incidence in the general population. RESULTS: 1526 cases were reported, and the cumulative incidence was 3,17% and 0,48% in within-school acquired cases. 20,9% were vaccinated. During follow-up, there was an important change in incidence from weeks 49/21 on, at the time Omicron began to appear in Spain, with an increase in secondary cases, mostly high school students. We found a high correlation between general population and schooled children's cumulative incidence (R2 = 0,59). CONCLUSIONS: SARS-CoV-2 transmission in school settings has been affected by new circulating variants. Two propagation scenarios were identified, and they were like the community propagation pattern. This supports the hypothesis that school settings reflect the transmission in the community. Reinforcement of preventive measures and surveillance would have a positive effect on school settings.

Unsupervised outlier detection applied to SARS-CoV-2 nucleotide sequences can identify sequences of common variants and other variants of interest.

As of June 2022, the GISAID database contains more than 11 million SARS-CoV-2 genomes, including several thousand nucleotide sequences for the most common variants such as delta or omicron. These SARS-CoV-2 strains have been collected from patients around the world since the beginning of the pandemic. We start by assessing the similarity of all pairs of nucleotide sequences using the Jaccard index and principal component analysis. As shown previously in the literature, an unsupervised cluster analysis applied to the SARS-CoV-2 genomes results in clusters of sequences according to certain characteristics such as their strain or their clade. Importantly, we observe that nucleotide sequences of common variants are often outliers in clusters of sequences stemming from variants identified earlier on during the pandemic. Motivated by this finding, we are interested in applying outlier detection to nucleotide sequences. We demonstrate that nucleotide sequences of common variants (such as alpha, delta, or omicron) can be identified solely based on a statistical outlier criterion. We argue that outlier detection might be a useful surveillance tool to identify emerging variants in real time as the pandemic progresses.

Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Vaccine Effectiveness.

The incidence and death toll due to SARS-CoV-2 infection varied time-to-time; and depended on several factors, including severity (viral load), immune status, age, gender, vaccination status, and presence of comorbidities. The RNA genome of SARS-CoV-2 has mutated and produced several variants, which were classified by the SARS-CoV-2 Interagency Group (SIG) into four major categories. The first category; "Variant Being Monitored (VBM)", consists of Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Epsilon (B.1.427, B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621), and Zeta (P.2); the second category; "Variants of Concern" consists of Omicron (B.1.1.529). The third and fourth categories include "Variants of Interest (VOI)", and "Variants of High Consequence (VOHC)", respectively, and contain no variants classified currently under these categories. The surge in VBM and VOC poses a significant threat to public health globally as they exhibit altered virulence, transmissibility, diagnostic or therapeutic escape, and the ability to evade the host immune response. Studies have shown that certain mutations increase the infectivity and pathogenicity of the virus as demonstrated in the case of SARS-CoV-2, the Omicron variant. It is reported that the Omicron variant has >60 mutations with at least 30 mutations in the Spike protein ("S" protein) and 15 mutations in the receptor-binding domain (RBD), resulting in rapid attachment to target cells and immune evasion. The spread of VBM and VOCs has affected the actual protective efficacy of the first-generation vaccines (ChAdOx1, Ad26.COV2.S, NVX-CoV2373, BNT162b2). Currently, the data on the effectiveness of existing vaccines against newer variants of SARS-CoV-2 are very scanty; hence additional studies are immediately warranted. To this end, recent studies have initiated investigations to elucidate the structural features of crucial proteins of SARS-CoV-2 variants and their involvement in pathogenesis. In addition, intense research is in progress to develop better preventive and therapeutic strategies to halt the spread of COVID-19 caused by variants. This review summarizes the structure and life cycle of SARS-CoV-2, provides background information on several variants of SARS-CoV-2 and mutations associated with these variants, and reviews recent studies on the safety and efficacy of major vaccines/vaccine candidates approved against SARS-CoV-2, and its variants.

Identification of SARS-CoV-2 Variants of Concern Using Amplicon Next-Generation Sequencing.

COVID-19 is caused by SARS-CoV-2, several virulent variants of which have emerged since 2019. More than 529 million people have been infected, and at least 6 million have died. Our aim was to develop a fast, accurate, low-cost method for detecting and identifying newly emerging variants of concern (VOCs) that could pose a global threat. The 341-bp DNA sequence of a specific region of the SARS-CoV-2's spike protein was amplified by a one-step PCR on RNA samples from 46 patients. The product was sequenced using next-generation sequencing (NGS). DNA sequences from seven genomes, the original Wuhan isolate and six different representative variants obtained from the GISAID website, were used as references. Complete whole-genome sequences from local isolates were also obtained from the GISAID website, and their RNA was used for comparison. We used an amplicon-based NGS method (termed VOC-NGS) for genotyping and successfully identified all 46 samples. Fifteen (32.6%) were like the original isolate. Twenty-seven were VOCs: nine (19.5%) Alpha, eight (19%) Delta, six (14%) Beta, and four (8.7%) Omicron. Two were variants of interest (VOI): one (2%) Kappa and one (2%) Zeta. Two samples were mixtures of two variants, one of Alpha and Beta and one of Alpha and Delta. The Spearman correlation between whole-genome sequencing (WGS) and VOC-NGS was significant (P < 0.001) with perfect agreement (Kappa = 0.916) for 36/38 (94.7%) samples with VOC-NGS detecting all the known VOCs. Genotyping by VOC-NGS enables rapid screening of high-throughput clinical samples that includes the identification of VOCs and mixtures of variants, at lower cost than WGS. IMPORTANCE The manuscript described SARS-Cov-2 genotyping by VOC-NGS, which presents an ideal balance of accuracy, rapidity, and cost for detecting and globally tracking VOCs and some VOI of SARS-CoV-2. A large number of clinical samples can be tested together. Rapid introduction of new mutations at a specific site of the spike protein necessitates efficient strain detection and identification to enable choice of treatment and the application of vaccination, as well as planning public health policy.

Cross-species tropism and antigenic landscapes of circulating SARS-CoV-2 variants.

Mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) may alter viral host tropism and affect the activities of neutralizing antibodies. Here, we investigated 153 RBD mutants and 11 globally circulating variants of concern (VOCs) and variants of interest (VOIs) (including Omicron) for their antigenic changes and cross-species tropism in cells expressing 18 ACE2 orthologs. Several RBD mutations strengthened viral infectivity in cells expressing ACE2 orthologs of non-human animals, particularly those less susceptible to the ancestral strain. The mutations surrounding amino acids (aas) 439-448 and aa 484 are more likely to cause neutralization resistance. Strikingly, enhanced cross-species infection potential in the mouse and ferret, instead of the neutralization-escape scores of the mutations, account for the positive correlation with the cumulative prevalence of mutations in humans. These findings present insights for potential drivers of circulating SARS-CoV-2 variants and provide informative parameters for tracking and forecasting spreading mutations.

Advances in the development of therapeutic strategies against COVID-19 and perspectives in the drug design for emerging SARS-CoV-2 variants.

Since Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has challenged public health around the world. Currently, there is an urgent need to explore antiviral therapeutic targets and effective clinical drugs. In this study, we systematically summarized two main therapeutic strategies against COVID-19, namely drugs targeting the SARS-CoV-2 life cycle and SARS-CoV-2-induced inflammation in host cells. The development of above two strategies is implemented by repurposing drugs and exploring potential targets. A comprehensive summary of promising drugs, especially cytokine inhibitors, and traditional Chinese medicine (TCM), provides recommendations for clinicians as evidence-based medicine in the actual clinical COVID-19 treatment. Considering the emerging SARS-CoV-2 variants greatly impact the effectiveness of drugs and vaccines, we reviewed the appearance and details of SARS-CoV-2 variants for further perspectives in drug design, which brings updating clues to develop therapeutical agents against the variants. Based on this, the development of broadly antiviral drugs, combined with immunomodulatory, or holistic therapy in the host, is prior to being considered for therapeutic interventions on mutant strains of SARS-CoV-2. Therefore, it is highly acclaimed the requirements of the concerted efforts from multi-disciplinary basic studies and clinical trials, which improves the accurate treatment of COVID-19 and optimizes the contingency measures to emerging SARS-CoV-2 variants.

The early SARS-CoV-2 epidemic in Senegal was driven by the local emergence of B.1.416 and the introduction of B.1.1.420 from Europe.

Molecular surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is growing in west Africa, especially in the Republic of Senegal. Here, we present a molecular epidemiology study of the early waves of SARS-CoV-2 infections in this country based on Bayesian phylogeographic approaches. Whereas the first wave in mid-2020 was characterized by a significant diversification of lineages and predominance of B.1.416, the second wave in late 2020 was composed primarily of B.1.1.420. Our results indicate that B.1.416 originated in Senegal and was exported mainly to Europe. In contrast, B.1.1.420 was introduced from Italy, gained fitness in Senegal, and then spread worldwide. Since both B.1.416 and B.1.1.420 lineages carry several positive selected mutations in the spike and nucleocapsid genes, each of which may explain their local dominance, their mutation profiles should be carefully monitored. As the pandemic continues to evolve, molecular surveillance in all regions of Africa will play a key role in stemming its spread.

Assessment of Mutations Associated With Genomic Variants of SARS-CoV-2: RT-qPCR as a Rapid and Affordable Tool to Monitoring Known Circulating Variants in Chile, 2021.

Since the first report of SARS-CoV-2 infection in humans, the virus has mutated to develop new viral variants with higher infection rates and more resistance to neutralization by antibodies elicited after natural SARS-CoV-2 infection or by vaccines. Therefore, rapid identification of viral variants circulating in the population is crucial for epidemiological assessment and efforts to contain the resurgence of the pandemic. Between January and November 2021, we performed a large variant RT-qPCR-based screening of mutations in the spike protein of 1851 SARS-CoV-2-positive samples derived from outpatients from the UC-Christus Health Network in Chile. In a portion of samples (n = 636), we validated our RT-qPCR-pipeline by WGS, obtaining a 99.2% concordance. Our results indicate that from January to March 2021 there was a dominance of non-identifiable variants by the RT-qPCR-based screening; however, throughout WGS we were able to identify the Lambda (C.37) variant of interest (VOI). From March to July, we observed the rapid emergence of mutations associated with the Gamma variant (P.1), which was quickly replaced by the appearance of a combination of samples harboring mutations associated with the Delta variant (B.1.617.2), which predominated until the end of the study. Our results highlight the applicability of cost-effective RT-qPCR-based screening of mutations associated with known variants of concern (VOC), VOI and variants under monitoring (VUM) of SARS-CoV-2, being a rapid and reliable tool that complements WGS-based surveillance.

Introduction of SARS-CoV-2 C.37 (WHO VOI lambda) in the Sao Paulo State, Southeast Brazil.

The Lambda variants of interest (VOI) (C37/GR/452Q.V1/21G) was initially reported in Lima, Peru but has gained rapid dissemination through other Latin American countries. Nevertheless, the dissemination and molecular epidemiology of the Lambda VOI in Brazil is unknown apart from a single case report. In this respect, we characterized the circulation of the SARS-CoV-2 Lambda VOI (C37/GR/452Q.V1/21G) in Sao Paulo State, Brazil. From March to June 2021, we identified seven Lambda isolates in a set of approximately 8000 newly sequenced genomes of the Network for Pandemic Alert of Emerging SARS-CoV-2 variants from Sao Paulo State. Interestingly, in three of the positive patients, the Lambda VOI infection was probably related to a contact transmission. These individuals were fully vaccinated to COVID-19 and presented mild symptoms. The remaining positive for Lambda VOI individuals showed different levels of COVID-19 symptoms and one of them needed hospitalization (score 5, WHO). In our study, we present a low level of Lambda VOI circulation in the Sao Paulo State. This reinforces the essential role of molecular surveillance for the effective SARS-CoV-2 pandemic response, especially in regard to circulating variants.

Spatial and temporal distribution of SARS-CoV-2 diversity circulating in wastewater.

Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.7 lineage and the ability of the technique to anticipate the detection of certain mutations before they are detected in clinical samples. The study proves the usefulness of sewage sequencing to track Variants of Concern that can complement clinical testing to help in decision-making and in the analysis of the evolution of the pandemic.

Antigenicity of the Mu (B.1.621) and A.2.5 SARS-CoV-2 Spikes.

The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Here, we assessed ACE2 binding and antigenicity of Mu (B.1.621) and A.2.5 Spikes. Both these variants carry some mutations shared by other emerging variants. Some of the pivotal mutations such as N501Y and E484K in the receptor-binding domain (RBD) detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is present in A.2.5. In this study, we observed that these Spike variants bound better to the ACE2 receptor in a temperature-dependent manner. Pseudoviral particles bearing the Spike of Mu were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta (B.1.351) and Delta (B.1.617.2) Spikes. Altogether, our results indicate the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization ability of each emerging variant.

A comprehensive overview of identified mutations in SARS CoV-2 spike glycoprotein among Iranian patients.

Since late 2019, when SARS-CoV-2 was reported at Wuhan, several sequence analyses have been performed and SARS-CoV-2 genome sequences have been submitted in various databases. Moreover, the impact of these variants on infectivity and response to neutralizing antibodies has been assessed. In the present study, we retrieved a total number of 176 complete and high-quality S glycoprotein sequences of Iranian SARS-COV-2 in public database of the GISAID and GenBank from April 2020 up to May 2021. Then, we identified the number of variables, singleton and parsimony informative sites at both gene and protein levels and discussed the possible functional consequences of important mutations on the infectivity and response to neutralizing antibodies. Phylogenetic tree was constructed to represent the relationship between Iranian SARS-COV2 and variants of concern (VOC), variants of interest (VOI) and reference sequence. We found that the four current VOCs - Alpha, Beta, Gamma and Delta - are circulated in different regions in Iran. The Delta variant is notably more transmissible than other variants, and is expected to become a dominant variant. However, some of the Delta variants in Iran carry an additional mutation, namely E1202Q in the HR2 subdomain that might confer an advantage to viral/cell membrane fusion process. We also observed some more common mutations such as an N-terminal domain (NTD) deletion at position I210 and P863H in fusion peptide-heptad repeat 1 span region in Iranian SARS-COV-2. The reported mutations in the current project have practical significance in prediction of disease spread as well as design of vaccines and drugs.

SARS-CoV-2 variant detection with ADSSpike.

The SARS-CoV-2 coronavirus pandemic has been an unprecedented challenge to global pandemic response and preparedness. With the continuous appearance of new SARS-CoV-2 variants, it is imperative to implement tools for genomic surveillance and diagnosis in order to decrease viral transmission and prevalence. The ADSSpike workflow was developed with the goal of identifying signature SNPs from the S gene associated with SARS-CoV-2 variants through amplicon deep sequencing. Seventy-two samples were sequenced, and 30 mutations were identified. Among those, signature SNPs were linked to 2 Zeta-VOI (P.2) samples and one to the Alpha-VOC (B.1.17). An average depth of 700 reads was found to properlycorrectly identify all SNPs and deletions pertinent to SARS-CoV-2 mutants. ADSSpike is the first workflow to provide a practical, cost-effective, and scalable solution to diagnose SARS-CoV-2 VOC/VOI in the clinical laboratory, adding a valuable tool to public health measures to fight the COVID-19 pandemic for approximately $41.85 USD/reaction.

Tracking SARS-CoV-2 Spike Protein Mutations in the United States (January 2020-March 2021) Using a Statistical Learning Strategy.

The emergence and establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of interest (VOIs) and variants of concern (VOCs) highlight the importance of genomic surveillance. We propose a statistical learning strategy (SLS) for identifying and spatiotemporally tracking potentially relevant Spike protein mutations. We analyzed 167,893 Spike protein sequences from coronavirus disease 2019 (COVID-19) cases in the United States (excluding 21,391 sequences from VOI/VOC strains) deposited at GISAID from 19 January 2020 to 15 March 2021. Alignment against the reference Spike protein sequence led to the identification of viral residue variants (VRVs), i.e., residues harboring a substitution compared to the reference strain. Next, generalized additive models were applied to model VRV temporal dynamics and to identify VRVs with significant and substantial dynamics (false discovery rate q-value < 0.01; maximum VRV proportion >10% on at least one day). Unsupervised learning was then applied to hierarchically organize VRVs by spatiotemporal patterns and identify VRV-haplotypes. Finally, homology modeling was performed to gain insight into the potential impact of VRVs on Spike protein structure. We identified 90 VRVs, 71 of which had not previously been observed in a VOI/VOC, and 35 of which have emerged recently and are durably present. Our analysis identified 17 VRVs ~91 days earlier than their first corresponding VOI/VOC publication. Unsupervised learning revealed eight VRV-haplotypes of four VRVs or more, suggesting two emerging strains (B1.1.222 and B.1.234). Structural modeling supported a potential functional impact of the D1118H and L452R mutations. The SLS approach equally monitors all Spike residues over time, independently of existing phylogenic classifications, and is complementary to existing genomic surveillance methods.

Strong humoral immune responses against SARS-CoV-2 Spike after BNT162b2 mRNA vaccination with a 16-week interval between doses.

The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration.

A Routine Sanger Sequencing Target Specific Mutation Assay for SARS-CoV-2 Variants of Concern and Interest.

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have a potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein, along with certain mutations in other regions within the S protein as specific variants, in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.