A rapid and low-cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-based RT-qPCR.

BACKGROUND: The new SARS-CoV-2 variant VOC (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit). METHODS: To control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to the VOC (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, detected by using the Applied Biosystems TaqPath RT-PCR COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile. RESULTS: Our results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene. CONCLUSIONS: This technique may allow to identify patients carrying the VOC (202012/01) or a closely related variant, in case of shortage in sequencing.

High throughput diagnostics and dynamic risk assessment of SARS-CoV-2 variants of concern.

BACKGROUND: The rise of new SARS-CoV-2 variants worldwide requires global molecular surveillance strategies to support public health control. Early detection and evaluation of their associated risk of spreading within the population are pivotal. METHODS: Between April 2020 and February 2021, the UK Lighthouse Labs Network at Alderley Park tested more than eight million nose and throat swab samples for the presence of SARS-CoV-2, via PCR. The assay targeted three genomic regions of the virus: N, Orf1ab and S. Whole-genome next-generation sequencing was used to confirm positive PCR results. Positive results were mapped using the postal district origin of samples to allow real-time tracking of the spread of a new variant through the UK. FINDINGS: In mid-November 2020, the assay identified an increasing number of S gene negative, N and Orf1ab positive samples. Whole-genome sequencing demonstrated that the loss of S gene detection was due to the appearance of a SARS-CoV-2 lineage (B.1.1.7) designated as Variant of concern (VOC) 202012/01. By the beginning of January 2021, the new SARS-CoV-2 VOC comprised 70% of daily positive samples tested at Alderley Park and ∼98% by the end of February 2021. INTERPRETATION: The timeline view identified the rapid spread of the new SARS-CoV-2 variant across England during the first three weeks of December. Coupling high-throughput diagnostics and molecular surveillance was pivotal to the early detection of the spread of this variant. The availability of real-time tracking of an emerging variant is an important new tool to inform decision-making authorities for risk mitigation. In a respiratory pandemic, a tool for the timely response to the emergence and spread of a novel variant is vital, even more so when a variant is associated with the enhanced transmission, as has occurred with VOC 202012/01. FUNDING: None.

Discovery of Taroxaz-104: The first potent antidote of SARS-CoV-2 VOC-202012/01 strain.

Polyhydroxyphenols and nitrogenous heterocyclics are two of the most powerful active species of molecules in pharmaceutical chemistry, as each of them is renowned for its various bioactivities for humans. One of their outstanding actions is the antiviral activities, which clearly appear if the principal functional entities of both classes meet into one compound. The recent COVID-19 pandemic pushed us to computationally sift and assess our small library of synthetic 2-(3,4,5-trihydroxyphenyl)-1,3,4-oxadiazoles against the main coronaviral protein/enzymatic targets. Surprisingly, few ligands exhibited interesting low binding energies (strong inhibitory affinities) with some SARS-CoV-2 proteins, mainly the pivotal enzyme RNA-dependent RNA polymerase (nCoV-RdRp). One of these compounds was Taroxaz-104 (5,5'-{5,5'-[(1R,2R)-1,2-dihydroxyethane-1,2-diyl]bis(1,3,4-oxadiazole-5,2-diyl)}dibenzene-1,2,3-triol), which presented lower binding free energies of about -10.60 and -9.10 kcal/mol (as compared to the reference agent, GS-443902, which presented about -9.20 and -7.90 kcal/mol) with nCoV-RdRp-RNA and nCoV-RdRp alone, respectively. Extensive molecular modeling examination disclosed the potent Taroxaz-104 inhibition of one of the possible active/allosteric sites of nCoV-RdRp, since Taroxaz-104 molecule interacts with at least seven main amino acids of the presumed pocket/cavity of this nCoV-RdRp active site. The effective repurposing of Taroxaz-104 molecule was attained after the satisfactorily interesting results of the anti-COVID-19 bioassay were secured, since these data demonstrated that Taroxaz-104 showed very efficient anti-COVID-19 actions (anti-SARS-CoV-2 EC50 = 0.42 µM) with specific promising efficacy against the new SARS-CoV-2 strains. Additional research studies for the progress of Taroxaz-104 and other related polyphenolic 2,5-disubstituted-1,3,4-oxadiazole analogs as successful anti-SARS-CoV-2 medications, via, e.g., preclinical/clinical trials, are pressingly required.

Comparison of SARS-CoV-2 Variants of Concern 202012/01 (U.K. Variant) and D614G Variant Transmission by Different Routes in Syrian Hamsters.

Introduction: Many SARS-CoV-2 variants of concern (VOC) have been reported recently that were linked to increased transmission. In our earlier study using VOC 202012/01 (U.K. variant) and D614G variant in the hamster model, we observed higher viral RNA shedding through nasal wash in the case of U.K. variant with lower pathogenicity in lung. In this study, we have studied transmission of these two variants by direct contact, aerosol, and fomite routes in Syrian hamsters and compared the viral load and body weight changes in hamsters exposed by both variants to understand the transmission efficiency. Methods: Nasal, throat, and rectal swabs were collected sequentially to assess viral load till 14 days. Results: Transmission could be established by direct, aerosol, and fomite contact in Syrian hamsters. Body weight loss or viral load in the contact animals exposed did not show any statistical significance. Conclusion: The study demonstrated comparable transmission of both U.K. and D614G variants of SARS-CoV-2 in Syrian hamsters in the given conditions. Provided these data, it seems that all the routes of exposure are effective leading to higher transmission.

Modeling the impact of SARS-CoV-2 variants and vaccines on the spread of COVID-19.

The continuous mutation of SARS-CoV-2 opens the possibility of the appearance of new variants of the virus with important differences in its spreading characteristics, mortality rates, etc. On 14 December 2020, the United Kingdom reported a potentially more contagious coronavirus variant, present in that country, which is referred to as VOC 202012/01. On 18 December 2020, the South African government also announced the emergence of a new variant in a scenario similar to that of the UK, which is referred to as variant 501.V2. Another important milestone regarding this pandemic was the beginning, in December 2020, of vaccination campaigns in several countries. There are several vaccines, with different characteristics, developed by various laboratories and research centers. A natural question arises: what could be the impact of these variants and vaccines on the spread of COVID-19? Many models have been proposed to simulate the spread of COVID-19 but, to the best of our knowledge, none of them incorporates the effects of potential SARS-CoV-2 variants together with the vaccines in the spread of COVID-19. We develop here a θ - i j -SVEIHQRD mathematical model able to simulate the possible impact of this type of variants and of the vaccines, together with the main mechanisms influencing the disease spread. The model may be of interest for policy makers, as a tool to evaluate different possible future scenarios. We apply the model to the particular case of Italy (as an example of study case), showing different outcomes. We observe that the vaccines may reduce the infections, but they might not be enough for avoiding a new wave, with the current expected vaccination rates in that country, if the control measures are relaxed. Furthermore, a more contagious variant could increase significantly the cases, becoming the most common way of infection. We show how, even with the pandemic cases slowing down (with an effective reproduction number less than 1) and the disease seeming to be under control, the effective reproduction number of just the new variant may be greater than 1 and, eventually, the number of infections would increase towards a new disease wave. Therefore, a rigorous follow-up of the evolution of the number of infections with any potentially more dangerous new variant is of paramount importance at any stage of the pandemic.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Investigation of an outbreak of symptomatic SARS-CoV-2 VOC 202012/01-lineage B.1.1.7 infection in healthcare workers, Italy.

OBJECTIVES: In December 2020, Italy began a national immunization campaign using the BNT162b2 coronavirus disease 2019 (COVID-19) mRNA vaccine, prioritizing healthcare workers (HCWs). Immune serum from vaccinated subjects seems (largely) to retain titres of neutralizing antibodies, even against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VOC 202012/01-lineage B.1.1.7. Here, we describe an outbreak of SARS-CoV-2 lineage B.1.1.7 infection in three HCWs in a hospital setting; two of the HCWs were fully vaccinated (i.e. had received two doses). METHODS: Two physicians and one nurse working on the same shift on 20th February 2021 were involved in the outbreak. Real-time PCR, antigen tests, and serological tests for the IgG anti-spike protein of SARS-CoV-2 were performed, along with whole-genome sequencing (WGS). RESULTS: SARS-CoV-2 infection was confirmed in all three HCWs; all presented with mild symptoms of COVID-19. The two physicians were fully vaccinated with BNT162b2 vaccine, with the second dose administered 1 month before symptom onset. Both had high titres of IgG anti-spike antibodies at the time of diagnosis. WGS confirmed that all virus strains were VOC 202012/01-lineage B.1.1.7, suggesting a common source of exposure. Epidemiological investigation revealed that the suspected source was a SARS-CoV-2-positive patient who required endotracheal intubation due to severe COVID-19. All procedures were carried out using a full suite of personal protective equipment (PPE). CONCLUSIONS: This mini-outbreak highlights some important issues about the efficacy of vaccines against transmission of SARS-CoV-2 variants, the high risk of exposure among HCWs, and the need for optimized implementation of PPE in hospitals. The wide circulation of VOC 202012/01 in Europe and Italy highlights the need to improve surveillance and genetic sequencing.

Rapid Spread of the SARS-CoV-2 Variant of Concern 202012/01 in Southern Italy (December 2020-March 2021).

Epidemiological and virological studies have revealed that SARS-CoV-2 variants of concern (VOCs) are emerging globally, including in Europe. The aim of this study was to evaluate the spread of B.1.1.7-lineage SARS-CoV-2 in southern Italy from December 2020-March 2021 through the detection of the S gene target failure (SGTF), which could be considered a robust proxy of VOC B.1.1.7. SGTF was assessed on 3075 samples from week 52/2020 to week 10/2021. A subset of positive samples identified in the Apulia region during the study period was subjected to whole-genome sequencing (WGS). A descriptive and statistical analysis of the demographic and clinical characteristics of cases according to SGTF status was performed. Overall, 20.2% of samples showed SGTF; 155 strains were confirmed as VOC 202012/01 by WGS. The proportion of SGTF-positive samples rapidly increased over time, reaching 69.2% in week 10/2021. SGTF-positive cases were more likely to be symptomatic and to result in hospitalization (p < 0.0001). Despite the implementation of large-scale non-pharmaceutical interventions (NPIs), such as the closure of schools and local lockdowns, a rapid spread of VOC 202012/01 was observed in southern Italy. Strengthened NPIs and rapid vaccine deployment, first among priority groups and then among the general population, are crucial both to contain the spread of VOC 202012/01 and to flatten the curve of the third wave.

Short Communication:Evidence of Novel SARS-CoV-2 Variants Circulation in Romania.

New SARS-CoV-2 variants are constantly emerging and putting a strain on public health systems by spreading faster and potentially evading immune protection through vaccination. One of these strains is the B.1.1.7 variant that has initially been described in the United Kingdom and has subsequently spread to several countries. Monitoring the amplification of the S gene-a major hotspot for molecular evolution-by reverse transcription polymerase chain reaction (RT-PCR) allows rapidly screening for such variants. This report describes the detection of sequence variants in Romania by using this strategy followed by next-generation sequencing of the entire genome for confirmation and further characterization. One B.1.1.7 and three B.1.258 sequences were confirmed. Each of these strains presented additional mutations with possible impact on the replicative capacity. Public health strategies should be devised to ensure molecular monitoring of SARS-CoV-2 evolution during the pandemic and allow adequate and rapid reaction.

The Genetic Variant of SARS-CoV-2: would It Matter for Controlling the Devastating Pandemic?

The pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is far from being controlled despite the great effort that have been taken throughout the world. Herd immunity through vaccination is our major expectation to rein the virus. However, the emergence of widespread genetic variants could potentially undermine the vaccines. The evidence that some variants could evade immune responses elicited by vaccines and previous infection is growing. In this review, we summarized the current understanding on five notable genetic variants, i.e., D614G, Cluster 5, VOC 202012/01, 501Y.V2 and P.1, and discussed the potential impact of these variants on the virus transmission, pathogenesis and vaccine efficacy. We also highlight that mutations in the N-terminal domain of spike protein should be considered when evaluating the antibody neutralization abilities. Among these genetic variants, a concern of genetic variant 501Y.V2 to escape the protection by vaccines was raised. We therefore call for new vaccines targeting this variant to be developed.

Potent toxic effects of Taroxaz-104 on the replication of SARS-CoV-2 particles.

Polyphenolics and 1,3,4-oxadiazoles are two of the most potent bioactive classes of compounds in medicinal chemistry, since both are known for their diverse pharmacological activities in humans. One of their prominent activities is the antimicrobial/antiviral activities, which are much apparent when the key functional structural moieties of both of them meet into the same compounds. The current COVID-19 pandemic motivated us to computationally screen and evaluate our library of previously-synthesized 2-(3,4,5-trihydroxyphenyl)-1,3,4-oxadiazoles against the major SARS-CoV-2 protein targets. Interestingly, few ligands showed promising low binding free energies (potent inhibitory interactions/affinities) with the active sites of some coronaviral-2 enzymes, specially the RNA-dependent RNA polymerase (nCoV-RdRp). One of them was 5,5'-{5,5'-[(1R,2R)-1,2-dihydroxyethane-1,2-diyl]bis(1,3,4-oxadiazole-5,2-diyl)}dibenzene-1,2,3-triol (Taroxaz-104), which showed significantly low binding energies (-10.60 and -9.10 kcal/mol) with nCoV-RdRp-RNA and nCoV-RdRp alone, respectively. These binding energies are even considerably lower than those of remdesivir potent active metabolite GS-443902 (which showed -9.20 and -7.90 kcal/mol with the same targets, respectively). Further computational molecular investigation revealed that Taroxaz-104 molecule strongly inhibits one of the potential active sites of nCoV-RdRp (the one with which GS-443902 molecule mainly interacts), since it interacts with at least seven major active amino acid residues of its predicted pocket. The successful repurposing of Taroxaz-104 has been achieved after the promising results of the anti-COVID-19 biological assay were obtained, as the data showed that Taroxaz-104 exhibited very significant anti-COVID-19 activities (anti-SARS-CoV-2 EC50 = 0.42 µM) with interesting effectiveness against the new strains/variants of SARS-CoV-2. Further investigations for the development of Taroxaz-104 and its coming polyphenolic 2,5-disubstituted-1,3,4-oxadiazole derivatives as anti-COVID-19 drugs, through in vivo bioevaluations and clinical trials research, are urgently needed.

Emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States.

The highly transmissible B.1.1.7 variant of SARS-CoV-2, first identified in the United Kingdom, has gained a foothold across the world. Using S gene target failure (SGTF) and SARS-CoV-2 genomic sequencing, we investigated the prevalence and dynamics of this variant in the United States (US), tracking it back to its early emergence. We found that, while the fraction of B.1.1.7 varied by state, the variant increased at a logistic rate with a roughly weekly doubling rate and an increased transmission of 40%-50%. We revealed several independent introductions of B.1.1.7 into the US as early as late November 2020, with community transmission spreading it to most states within months. We show that the US is on a similar trajectory as other countries where B.1.1.7 became dominant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

SARS-CoV-2 Variant of Concern 202012/01 Has about Twofold Replicative Advantage and Acquires Concerning Mutations.

The novel SARS-CoV-2 Variant of Concern (VOC)-202012/01 (also known as B.1.1.7), first collected in United Kingdom on 20 September 2020, is a rapidly growing lineage that in January 2021 constituted 86% of all SARS-CoV-2 genomes sequenced in England. The VOC has been detected in 40 out of 46 countries that reported at least 50 genomes in January 2021. We have estimated that the replicative advantage of the VOC is in the range 1.83-2.18 [95% CI: 1.71-2.40] with respect to the 20A.EU1 variant that dominated in England in November 2020, and in range 1.65-1.72 [95% CI: 1.46-2.04] in Wales, Scotland, Denmark, and USA. As the VOC strain will likely spread globally towards fixation, it is important to monitor its molecular evolution. We have estimated growth rates of expanding mutations acquired by the VOC lineage to find that the L18F substitution in spike has initiated a fast growing VOC substrain. The L18F substitution is of significance because it has been found to compromise binding of neutralizing antibodies. Of concern are immune escape mutations acquired by the VOC: E484K, F490S, S494P (in the receptor binding motif of spike) and Q677H, Q675H (in the proximity of the polybasic cleavage site at the S1/S2 boundary). These mutants may hinder efficiency of existing vaccines and expand in response to the increasing after-infection or vaccine-induced seroprevalence.

Infection sustained by lineage B.1.1.7 of SARS-CoV-2 is characterised by longer persistence and higher viral RNA loads in nasopharyngeal swabs.

Following the announcement on December 2020 about the emergence of a new variant (VOC 202012/ 01, B.1.1.7 lineage) in the United Kingdom, a targeted surveillance was put in place in the Abruzzo region (Italy), which allowed detection of 313 persons affected by lineage B.1.1.7, up to the 20th of February 2021. We investigated the results of RT-PCR on nasopharyngeal swabs tested from December 2020 to February 2021 to verify any difference on the viral load and persistence between people infected by lineage B.1.1.7 and others. Statistically significant lower values of CT associated with the detection of the N protein encoding gene (CT N) were observed in persons with lineage B.1.1.7 infection (median CT N = 15.8)in comparison to those infected by other lineages (median CT N = 16.9). A significantly longer duration of the persistence of SARS-CoV-2 RNA in nasopharyngeal swabs was observed in persons with lineage B.1.1.7 infection (16 days) in comparison to those infected by other lineages (14 days).

SARS-CoV-2 variants combining spike mutations and the absence of ORF8 may be more transmissible and require close monitoring.

The SARS-CoV-2 Variant of Concern 202012/01 (VOC-202012/01) emerged in southeast England and rapidly spread worldwide. This variant is believed to be more transmissible, with all attention being given to its spike mutations. However, VOC-202012/01 has also a mutation (Q27stop) that truncates the ORF8, a likely immune evasion protein. Removal of ORF8 changes the clinical outset of the disease, which may affect the virus transmissibility. Here I provide a detailed analysis of all reported ORF8-deficient lineages found in the background of relevant spike mutations, identified among 231,433 SARS-CoV-2 genomes. I found 19 ORF8 nonsense mutations, most of them occurring in the 5' half of the gene. The ORF8-deficient lineages were rare, representing 0.67% of sequenced genomes. Nevertheless, I identified two clusters of related sequences that emerged recently and spread in different countries. The widespread D614G spike mutation was found in most ORF-deficient lineages. Although less frequent, HV69-70del and L5F spike mutations occurred in the background of six different ORF8 nonsense mutations. I also confirmed that VOC-202012/01 is the ORF8-deficient variant with more spike mutations reported to date, although other variants could have up to six spike mutations, some of putative biological relevance. Overall, these results suggest that monitoring ORF8-deficient lineages is important for the progression of the COVID-19 pandemic, particularly when associated with relevant spike mutations.

Analysing the impact of global demographic characteristics over the COVID-19 spread using class rule mining and pattern matching.

Since the coronavirus disease (COVID-19) outbreak in December 2019, studies have been addressing diverse aspects in relation to COVID-19 and Variant of Concern 202012/01 (VOC 202012/01) such as potential symptoms and predictive tools. However, limited work has been performed towards the modelling of complex associations between the combined demographic attributes and varying nature of the COVID-19 infections across the globe. This study presents an intelligent approach to investigate the multi-dimensional associations between demographic attributes and COVID-19 global variations. We gather multiple demographic attributes and COVID-19 infection data (by 8 January 2021) from reliable sources, which are then processed by intelligent algorithms to identify the significant associations and patterns within the data. Statistical results and experts' reports indicate strong associations between COVID-19 severity levels across the globe and certain demographic attributes, e.g. female smokers, when combined together with other attributes. The outcomes will aid the understanding of the dynamics of disease spread and its progression, which in turn may support policy makers, medical specialists and society, in better understanding and effective management of the disease.

VOC 202012/01 Variant Is Effectively Neutralized by Antibodies Produced by Patients Infected before Its Diffusion in Italy.

The coronavirus disease 2019 (Covid-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and presents a global health emergency that needs urgent intervention. Viruses constantly change through mutation, and new variants of a virus are expected to occur over time. In the United Kingdom (UK), a new variant called B.1.1.7 has emerged with an unusually large number of mutations. The aim of this study is to evaluate the level of protection of sera from 12 patients infected and later healed in Apulia Region (Italy) with Covid-19 between March and November 2020, when the English variant was not circulating in this territory yet, against the new VOC 202012/01 variant by seroneutralization assay. The sera of patients had already been tested before, using a virus belonging to the lineage B.1 and showed an antibody neutralizing titer ranging between 1:160 and 1:320. All the 12 sera donors confirmed the same titers of neutralizing antibodies obtained with a strain belonging to the lineage B.1.1.7 (VOC 202012/01). These data indicate that antibodies produced in subjects infected with variants of Sars-CoV-2 strain before the appearance of the English one, seem to have a neutralizing power also against this variant.

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom, October to November 2020.

Two new SARS-CoV-2 lineages with the N501Y mutation in the receptor-binding domain of the spike protein spread rapidly in the United Kingdom. We estimated that the earlier 501Y lineage without amino acid deletion Δ69/Δ70, circulating mainly between early September and mid-November, was 10% (6-13%) more transmissible than the 501N lineage, and the 501Y lineage with amino acid deletion Δ69/Δ70, circulating since late September, was 75% (70-80%) more transmissible than the 501N lineage.