Genome sequencing of SARS-CoV-2 reveals the prevalence of variant B 1.1.7 in Egypt

Recently, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in Coronavirus Disease 2019 (COVID-19) outbreak. A new SARS-CoV-2 strain is expected to emerge in late 2020, including B.1.1.7. The high transmission rate of SARS-CoV-2 B.1.1.7 has raised public health concerns in several countries. Hence, in this study, we assessed the sequencing of SARS-COV2 to reveals the prevalence of the SARS-CoV-2 Alpha variant (B 1.1.7) in Egypt. We found that the viral transmission of the alpha variant is expanding. Moreover, based on hospitalizations and case fatality rates, there is a potential for increasing severity. There was no effect on susceptibility to Emergency Use Authorization monoclonal antibody treatments. However, there was minimal impact on neutralization by convalescent and post-vaccination sera. Samples have been clustered into the 20D sub clade for the majority of them. The eight samples shown in our study are considered the first recorded samples with the Alpha variant in Egypt. Therefore, The Egyptian government, represented by the Ministry of Health, must take all measures to examine the compatibility of the currently used vaccines with this new strain and the feasibility of the treatment protocol presently used with such strains developed in the Arab Republic of Egypt.


Introduction
Recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 2 resulted in Coronavirus Disease 2019 (COVID-19) outbreak. There have been more than 260 million cases of COVID-19 pandemic since the outbreak, with the mortality impact at roughly 3.9 million (Jamwal et al., 2021). Controlling COVID-19 can be enhanced by vaccination, separating from others socially, general cleanliness, and a large number of diagnostic tests Ferretti et al., 2020).
The genome of SARS-CoV-2 has been estimated to be 29.9 kb (Lu et al., 2020). It is composed of a nucleocapsid protein (N) and an envelope. Envelopes are related with three types of structural proteins: spike protein (S), membrane protein (M), and envelope protein (E). N, S, M, and E are four structural proteins in the SARS-CoV-2 genome, although there are total of 16 non-structural proteins (NSP1-16). Also known as NSP 12, RdRp which plays a key part in the proliferation of (Naqvi et al., 2020).
SARS-CoV-2 was found to be transmissible in roughly 55% of cases, with approximately 30% of people remain asymptomatic . Spike protein receptor binding domain alterations appear to be responsible for the increased transmissibility of novel variations (Cherian et al., 2021;Funk et al., 2021). A significant number of people must be tested in order to detect SARS-CoV-2 infections and, more critically, to identify asymptomatic carriers who may unknowingly infect a huge number of people (Lu et al., 2020). A new SARS-CoV-2 strain is expected to emerge in late 2020, and world health organization (WHO) has classified it as a Variant of Concern or VOI (VOCs). As of now, VOCs include Alpha, Beta, Gamma, Delta, and Epsilon, while VOIs include Epsilon, Zeta, Eta, Theta and Kappa and B.1.427/B.1.429 (Epsilon) (WHO, 2021). There has been viral evolution since the discovery of SARS-CoV-2, however these VOCs have increased transmissibility and severity as well as altered antigenicity, which could have consequences for acquired immunity or efficiency of current vaccinations (Collier et al., 2021;Challen et al., 2021;Madhi et al., 2021;Wang et al., 2021).
The high transmission rate of SARS-CoV-2 B.1.1.7 in the United Kingdom (UK) has raised public health concerns. In addition to the D614G mutation, this variation features eight additional nonsynonymous variants in spike: H69-V70, Y144, N501Y, A570D, P681H, T716I, and D1118H. At least three B.1.1.7 spike mutations should be of concern, including the two amino acid deletion in the Nterminal domain (NTD), as well as the N501Y mutation in the receptor binding motif (RBM) and P681H mutation at the Furin cleavage site (P681H) ( et al., 2021). Interestingly, each of these three mutations can be found in other variants of interest, as well. Mathematical modeling data and epidemiological evidence suggest that this variant is more transmissible than the SARS-CoV-2 variants that were in circulation prior to its introduction; evidence of an increased mortality rate has also been reported, despite initial reports that it was not more pathogenic (England, 2021;Volz et al., 2020).
The first case detected in Egypt with the Alpha variant was recorded in March 2021 (GISAID, 2021). Hence, in this study, we assessed the sequencing of SARS-CoV-2 to reveal the prevalence of SARS-CoV-2 variant B 1.1.7 in Egypt.

Sampling
Oropharyngeal swabs (n = 1235) were taken from patients with suspected COVID-19 in fever hospital of army forces in Egypt. The samples were transported under biosafety requirements (transport bag 95Kpa) in viral transport medium Kit (Cat. no. A48498) at (2-4) C • to the biological prevention department at central chemical laboratories of Almaza in Cairo, Egypt.

Extraction of RNA and RT-PCR detection
Viral RNA was extracted using QIAamp Viral RNA Mini Kit (Cat. no. 52906). Then, samples were tested for three SARS-CoV-2 genes, i.e., ORF1ab and N protein, using the VIASURE kit (Cat. no.VS-NCO213H) according to the manufacturers' instructions. For each sample, we added 5 μL of extracted RNA to 15 μL of dehydrated master mix. Another tube for positive control was prepared by adding 5 μL of Positive Control to 15 μL of dehydrated master mix. The last tube was prepared for negative control by adding 5 μL of Negative Control to 15 μL of dehydrated master mix. Then, qPCR was performed on an Ariamx thermal cycler (Agilent, Germany), using the following conditions: 45 • C for 15 min, 95 • C for 2 min, 40 cycles of 95 • C for 10 s, and 60 • C for 50 s. Cycle thresholds (Ct) were analyzed using auto-analysis settings, with the threshold lines falling within the exponential phase of the fluorescence curves and above any background signal. We used HEX dye as a fluorescent label for each reaction to determine the Ct value for the internal positive control.

Library preparation and next-generation sequencing
According to manufacturers' instructions, library preparation was done using the AviSeq COV19 NGS Library Prep kit (Ref. AVG202096). Next-generation sequencing was done by using the Illumina machine (Iseq-100 instrument). RNA was measured on Qubit 2.0 Fluorometer by using Qubit™ RNA HS 100 Assay Kit (Cat.No.Q32852). Then, cDNA was prepared by adding RT Primer Mix DP (BATCH No.020402C). In addition, a multiplex PCR reaction was done to amplify the entire SARS-CoV-2 genome. Digestion was done to remove any nonspecific PCR products. After library preparation, the quality of the library and the amplification of viral RNA were visualized via gel electrophoresis and measured using DNA HS 100 Assay Kit (Cat.No.Q32852).

Data analysis
FASTA files were generated using Abiomix software. Then Nextclade Tool (version 1.5.2) was used for viral genome alignment, mutation calling, quality checks, phylogenetic placement, and clade assignment. Genome sequencing alignment was done by using Burrows-Wheeler Aligner (BWA) software. Basic Local Alignment Search Tool (BLAST) was used to find similarities between nucleotide and amino acid sequences.

Molecular identification
We used RT-PCR for molecular identification. From April 2020 to July 2021, we collected 1235 COVID-19 positive samples, but only a few hundred models were chosen for sequencing (Cq from 14 to 20).

Sequence data analysis and phylogenetic analysis
Whole Genome sequencing alignment using BWA software resulted in 99 successful high-quality genomes. These sequences were covered the entire genome of reference SARS-CoV-2 (RefSeq: NC_045512.2). Basic Local Alignment Search Tool (BLAST) was used to find similarities between nucleotide and amino acid sequences. Hence, nucleotide identity ranged from 99.8% to 100%, and amino acid identity ranged from 99.7% to 100%.
The genetic mutation using Nextclade Tool showed the viral genome, including ORF1a, ORF1b, ORF3a, ORF6, ORF7a, ORF7b, ORF8, S, E, M,       Fig. 1. These clades were shown in Rectangular and Radial Phylogeny as shown in Fig. 1A and Fig .1B respectively. The most common clade was 20D with 61.6%, and the Alpha variant was 8%. There were about seven sequences clustered as 19A with no mutation in S-gene. However, a single mutation with high quality in ten sequences was assigned as 20A. The clad 20A with D614G amino acids mutations in S-gene was shown in Table 4. Moreover, 11sequences were clustered as 19B with R102S, A292V, N501T, and H655Y amino acids mutations in S-gene. However, two sequences were clustered as 20B with N501T, D614G, and Q677H amino acids mutations in S-gene, as shown in Table 3.
Finally, eight sequences were clustered as 20I (Alpha, V1) with N501T, D614G, and Q677H amino acid mutations in S-gene, as shown in Table 1. These eight last sequences were considered the first UK strain detected in Egypt by April 2021 (GISAID, 2021).

Discussion
According to our study, we found that the viral transmission of the alpha variant is expanding. Moreover, based on hospitalizations and case fatality rates, there is a potential for increasing severity. There was no effect on susceptibility to Emergency Use Authorization (EUA) monoclonal antibody treatments (FDA, 2021). However, there was minimal impact on neutralization by convalescent and post-vaccination sera. Samples have been clustered into the 20D sub clade for the majority of them.
New SARS-CoV-2 variations have emerged in recent months, and their frequency in local populations and even worldwide has shifted fast and repeatedly (Hodcroft et al., 2020;Korber et al., 2020). A number of spike mutations have been found in newer variations, which raises the possibility of immunological escape (Naveca et al., 2021;Tegally et al., 2020).
From the First discovery in England in September 2020, the B.1.1.7 variety has already spread to over 70 countries (Hodcroft et al., 2020). After a mutation was found in the spike protein of a fast-spreading B.1.1.7 strain, it became 56% more transmissible than the earlier SARS-CoV2 strain (Davies et al., 2021a). In South Africa, a new fastspreading form of SARS-CoV-2 has been identified as B.1.351 virus. SARS-CoV-2 strains of lineage also were recently found in India: B.1.617.1 and B.1.617.2 (Funk et al., 2021). As a result of this new variation, existing control methods are likely to be less successful, and governments may need to take more aggressive efforts to attain the same level of control (Frank, 1996).
Vaccination was affected by the increase in VOC transmission. A rapid and efficient vaccination delivery system is therefore essential for reducing the effects of the pandemic as soon as possible. VOC will also increase herd immunity threshold, which means that future SARS-CoV-2 burden will be greater and more vaccination coverage will be necessary to obtain herd immunity (Davies et al., 2021a).
Finally, eight sequences were clustered as 20I (Alpha, V1) with N501T, D614G, and Q677H amino acid mutations in S-gene. These eight last sequences are considered the first UK strain detected in Egypt by April 2021.
When it comes to B.1.1.7/SGTF, B.1.351 and P.1, there is a higher risk of hospitalization and ICU admission in age groups 60 years in this study. As seen in Germany, B.1.1.7 dominance was associated with an increase in hospitalizations among those under 60 years of age (Funk et al., 2021;Lorbach, 2021). A greater infection rate in younger, schoolage age groups was documented in the UK before (Davies et al., 2021b;Gravagnuolo et al., 2021). Denmark has also documented higher hospitalization rates for B.1.1.7 patients (Bager et al., 2021). When exposed to convalescent sera, variation B.1.1.7 shows only a small reduction in neutralization susceptibility (1.5-fold average) (Muik et al., 2021). When persons receive both doses of both vaccines, neutralization titers have been reported to increase around 10-fold after the second dose, suggesting that a 2-fold decline in neutralization will have no influence on vaccine efficacy (Anderson et al., 2020;Walsh et al., 2020). Recently, it was discovered that the Novavax vaccination was 95,6% effective against the common variant and 85,6% against B.1.1.7 (Callaway and Mallapaty, 2021). In areas where the B.1.1.7 variety is prevalent, receiving the second dose as soon as possible is recommended for optimum efficacy (Shen et al., 2021).
A cautious strategy should be to experiment with variant sequences and conduct post-licensure investigations to detect changes in efficacy between the preexisting and new variants, according to vaccine developers if a vaccine's strain formulation is altered without any other modifications in its content. Licensing authorities may need to clarify the abbreviated marketing paths for the vaccine (Davies et al., 2021a).
Some of our findings have limitations. We can only assess how well the theories suggested are supported by the data. Still, there may be other viable explanations for the recurrence of cases that we did not investigate. Even though more experimental work could shed light on the biological mechanisms behind our observations, given our projections of a rapid future rise in incidence from the Alpha variant without additional control measures, we must consider what new approaches may be required to sufficiently reduce the ongoing transmission of SARS-CoV-2.

Conclusion
Viral transmission of the alpha variant is expanding. Moreover, based on hospitalizations and case fatality rates, there is a potential for increasing severity. There was no effect on susceptibility to Emergency Use Authorization monoclonal antibody treatments. However, there was minimal impact on neutralization by convalescent and post-vaccination sera. Samples have been clustered into the 20D sub clade for the majority of them. The eight samples shown in our study are considered the first recorded samples with Alpha variant in Egypt. Therefore, The Egyptian government, represented by the Ministry of Health, must take all measures to examine the compatibility of the currently used vaccines with this new strain and the feasibility of the treatment protocol presently used with such strains developed in the Arab Republic of Egypt.