Monitoring the spread of the SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) variants in the Moscow region using targeted high-throughput sequencing

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Abstract

Introduction. Since the outbreak of the COVID-19 pandemic caused by SARS-CoV-2 novel coronavirus, the international community has been concerned about the emergence of mutations altering some biological properties of the pathogen like increasing its infectivity or virulence. Particularly, since the end of 2020, several variants of concern have been identified around the world, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). However, the existing mechanism of detecting important mutations are not always effective enough, since only a relatively small part of all pathogen samples can be examined by whole genome sequencing due to its high cost.

Material and methods. In this study, we have designed special primer panel and used it for targeted highthroughput sequencing of several significant S-gene (spike) regions of SARS-CoV-2. The Illumina platform averaged approximately 50,000 paired-end reads with a length of ≥150 bp per sample. This method was used to examine 579 random samples obtained from COVID-19 patients in Moscow and the Moscow region from February to June 2021.

Results. This study demonstrated the dynamics of distribution of several SARS-CoV-2 strains and its some single mutations. It was found that the Delta strain appeared in the region in May 2021, and became prevalent in June, partially displacing other strains.

Discussion. The obtained results provide an opportunity to assign the viral samples to one of the strains, including the previously mentioned in time- and cost-effective manner. The approach can be used for standardization of the procedure of searching for mutations in individual regions of the SARS-CoV-2 genome. It allows to get a more detailed data about the epidemiological situation in a region.

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Introduction

Since it was first detected in Wuhan (the People’s Republic of China (PRC)) in December 2019 [1], the novel SARS-CoV-2 coronavirus has rapidly spread around the world and caused >4 million deaths [2]. Since the beginning of the pandemic, a number of effective therapeutic and preventive measures have been adopted to fight the novel coronavirus infection (COVID-19). They include using of therapeutic agents such as monoclonal antibodies (mAbs) [3][4] and vaccines [5–8] where the spike (S) protein of the virus acts as an antigen.

At the end of 2020, the global scientific community described several SARS-CoV-2 variants of concern and special attention. They are Alpha (formerly referred to as the British variant, B.1.1.7), Beta (South-African, B.1.351), Gamma (Brazilian, P.1) and Delta (Indian, B.1.617.2) variants. The above variants of the virus sparked the interest of researchers after a number of geographic regions of the world had reported an increase in the frequency of human-to-human transmission of these infectious agents; later, the new variants of the pathogen were detected in many countries. For example, the Alpha variant spread rapidly in southeast England, causing a sharp increase in COVID-19 cases; shortly after, it was detected in the United States (USA Centers for Disease Control and Prevention (CDC)) [9], having become the dominant variant circulating in the country by April 2021. Similarly, the variants from the Republic of South Africa and Brazil caused outbreaks of COVID-19 in these countries. The above variants of SARS-CoV-2 give rise to concern due to the E484K mutation in the S protein, which may reduce the efficacy of some therapeutic mAbs, hinder virus neutralization in vitro and cause the virus to evade the immune protection developed following the infection or vaccination [8][10–14].

In addition, 3 variants (Alpha, Beta and Gamma), in a key region of the S protein (the receptor-binding domain (RBD)), have the N501Y mutation associated with increased affinity for the angiotensin-converting enzyme 2 (ACE2) receptor. In its turn, it can contribute to increased transmissibility of the pathogen [15][16]. Specialists of the Central Research Institute for Epidemiology (CRIE) of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor) designed a reagent kit for fast detection of the above mutation in the viral genome by using loop-mediated isothermal amplification (LAMP) [17] to significantly reduce the number of samples required for whole genome sequencing aimed to detect and monitor new strains containing the N501Y mutation. However, the further emergence of similar strains, including strains with other mutations in the S protein gene, showed that LAMP-associated genomic substitutions at primer binding sites could decrease the effectiveness of the above technique. In addition, the detection and exploration of SARS-CoV-2 variants result in an extended list of mutation-caused changes of interest and subject to monitoring. For example, the Delta strain responsible for a sharp increase in the number of cases in India was detected in Russia in May 2021, and has been rapidly spreading in this country since that time. Furthermore, several local strains have been detected in Russia, including Siberian (B.1.1.397+) and Northwestern (B.1.1.370.1) strains carrying mutations in the S protein gene [18][19]. At the moment, the strains circulating in Russia are being studied and monitored [20]. For the foregoing reasons, scientists need more efficient and universal tools to identify multiple significant mutations within a single cycle of the laboratory test. Although whole genome sequencing of viruses is undoubtedly the most detailed technique of the genetic analysis [21], economically speaking, it may not be the best option among the advanced sequencing techniques. It may pose difficulties when used in the setting of steadily increasing COVID-19 cases, including repeat disease cases. In addition, whole genome identification may be impractical for effective epidemiological surveillance, considering that the most significant changes take place in a small part of the viral genome.

In this article, we describe the identification of isolates belonging to different SARS-CoV-2 variants (including Alpha, Beta and Delta strains) by using targeted high-throughput next-generation sequencing. We designed a primer panel intended for fast and efficient targeted amplification of genomic fragments, bypassing ligation of adapters when preparing samples for sequencing due to modification of oligonucleotides during the synthesis. The amplified regions include the most epidemiologically significant mutations corresponding to K417T, L452R, T478K, E484K, S494P, N501Y, A570D, P681H and other amino acid substitutions as well as to HV69-70 and Y144 deletions (Fig. 1). Therefore, not only the cost of sample preparation is significantly reduced, but also the amount of generated data is substantially reduced, making it possible to study a larger number of virus samples within a short period. The latter point is especially important in the situation when the frequency of different strains should be promptly assessed for the region or country of interest. Using our primer panel, we studied 579 random virus samples collected in Moscow and Moscow Region in February–June 2021, and found changes in frequencies of some mutations and strains in the above region.

Рис. 1. Локализация ампликонов, получаемых с помощью разработанной панели праймеров, относительно генома SARS-CoV-2.
Примечание. Указан ряд аминокислотных замен и делеций, покрываемых панелью. Amp 1–5 – ампликоны; S – S-белок; RBD – рецептор-связывающий домен.
Fig. 1. Localization of the amplicons obtained using the primer panel relative to the SARS-CoV-2 virus genome.
Note. Several amino acid substitutions and deletions covered by the panel are indicated. Amp 1–5, amplicons; S, S-protein; RBD, receptor binding domain.

Materials and methods

In our study, we used biological materials from nasopharyngeal swabs from patients with symptoms of novel coronavirus infection. The presence of SARSCoV-2 in these patients was confirmed with a real-time reverse-transcription polymerase chain reaction (rRTPCR) assay by using an AmpliSens Cov-Bat-FL reagent kit (AmpliSens, Russia). The study was conducted with the informed consent of the patients; the research protocol was approved by the CRIE Ethics Committee (Protocol No. 111 of December 22, 2020). The samples were placed in the transport medium (CRIE, Russia). The RNA was extracted from the clinical material with the help of a RIBO-prep reagent kit (AmpliSens); the reverse transcription was performed by using a REVERTA-L kit (AmpliSens). Only the clinical samples, in which the cycle threshold (Ct) did not exceed 20 in the PCR test, were selected.

For amplification in a T100 Thermal Cycler (BioRad, USA), we used PCR-mix-2 blue (AmpliSens) containing Taq-polymerase. Then, the PCR-products were cleaned from the reaction mixture by using AMPureXP beads (Beckman Coulter, USA). The temperature profile for the amplification was as follows: 1) denaturation at 95 ºC for 30 seconds; 2) 38 amplification cycles: 95 ºC – 30 sec, 60 ºC – 20 sec, 72 ºC – 60 sec; 3) final elongation at 72ºC for 3 min. Indexing was performed by using PCR-mix-2 blue and EvaGreen (Biotium, USA) as a dye and a QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific, USA). The indexing was conducted with index primers compatible with a Nextera XT Index Kit v2 (N7xx – 26 possible variants and S5xx – 18 variants; for more information see the Illumina Adapter Sequences, Document # 1000000002694 v16, April 2021, https://support-docs.illumina.com/SHARE/AdapterSeq/illumina-adapter-sequences.pdf). The double indexing improves the accuracy of sample identification and makes it possible to test a large number of samples at a time. The temperature profile of the indexing was: 1) 98 ºC – 30 sec; 2) 15 cycles: 98 ºC – 10 sec, 65 ºC – 1 min 15 sec. Then, the samples went through another clean-up from the reaction mixture. The DNA concentration was measured with a Qubit 4.0 fluorometer (ThermoFisher Scientific). Based on the concentration, the samples were pooled and the pool quality was evaluated by using an Agilent 2100 bioanalyzer (Agilent Technologies, USA). Denaturation and estimation of the loading volume were performed according to the manufacturer’s instructions.

The sequencing was conducted on the Illumina MiSeq platform (Illumina, USA) by using a MiSeq Reagent Kit v2 (PE 150 + 150 or PE 250 + 250 cycles) or a MiSeq Reagent Kit v3 (PE 300 + 300 cycles) as well as on the Illumina HiSeq platform by using a v2 Rapid SBS kit (PE 250 + 250 cycles). There were around 50 thousand reads per sample, which is equivalent to approximately 0.3% of the information obtained when using a MiSeq Reagent Kit v2 (PE 150 + 150) or approximately 0.1% – when using a MiSeq Reagent Kit v3 (PE 300 + 300 cycles) during sequencing. The above approach makes it possible to avoid excessive expenses associated with testing numerous samples, while the average coverage level for most of the samples is ≥2,000.

The primers for the panel for targeted amplification of fragments of the S protein gene were selected manually based on the available information about the known epidemiologically significant mutations and the information about conserved regions of the genome. The melting temperature of oligonucleotides and the level of interaction between them were estimated with a Multiple Primer Analyzer (ThermoFisher Scientific). The BLASTn program [22] was used to evaluate the specificity of each obtained sequence in reference to all known organisms (first of all, the human organism, the genetic material of which is best represented in the sample), thus eliminating any non-specific interaction between the primer and DNA regions of human and other organisms. A total of 5 pairs of oligonuc-leotides were obtained; they also contained additional adapter sequences required for time and cost reduction during sample preparation. The synthesis was performed at Syntol Research and Production Company, LLC (Russia). The structure of primers is shown in the Table. The lengths of amplicons were selected to provide overall coverage of the target regions during their high-throughput sequencing on Illumina MiSeq platforms with the help of MiSeq v2 reagent kits (300 cycles), v2 (500 cycles) and v3 (600 cycles) and on Illumina HiSeq platforms by using a v2 Rapid SBS kit (500 cycles).

Последовательности олигонуклеотидов в праймерной панели
Sequences of oligonucleotides in the primer panel


Примечание. Специфические фрагменты отделены от адаптеров символом «-».
Note. Specific fragments are separated from adapters with a «-» symbol.

To analyze the sequencing data, the obtained reads were aligned to the reference genome of the SARS-CoV-2 virus by using the BWA software [23]. The BBTools program [24] was used to trim the adapter sequences in the reads. The coverage of 5 reads was accepted as the minimum coverage. The GATK program was used to find genetic variants [25]. The obtained sequences were uploaded to the VGARus nucleotide sequence database for SARSCoV-2 and its mutations (https://genome.crie.ru/).

The VMD software was used for visualization of the S protein molecule and for the subsequent drawings [26]. We used a structural model of S protein (the Protein Data Bank (PDB) ID: 7CAB), which was received by using cryoelectron microscopy [27].

Results

The period, during which the study was conducted in the Moscow Region, coincided with the time when SARS-CoV-2 strains of concern started spreading all over the world, including Russia, contributing to the risk of new waves of disease cases in a number of countries.

While only a relatively small (~2%) proportion of the Alpha strain was found in the isolates obtained in February 2021, its frequency increased to ~20% in March, supporting the data on high contagiousness of the strain [28]. However, no further widespread occurrence of the strain was recorded; the frequency rate gradually decreased, reaching almost zero in the middle of June. Most likely, this decrease was caused by the Delta strain that spread to Russia in May 2021 and previously may have caused an increase in the number of cases and deaths among the population of India. By mid-June 2021, the prevalence of this strain increased to 70% in Moscow, and, according to the data not included in this study, it has been steadily increasing to >90%. In addition, at the end of June 2021, there were cases of infection with the Delta Plus strain containing the additional K417N substitution [29], which was previously found in the Beta strain and which is located in one of the SARS-CoV-2 genome regions amplifiable by using the new primer panel (Fig. 2).

Notably, no significant spread of the Beta variant has been recorded in Moscow, though in April 2021, its proportion quickly increased to 13%, causing concern among the population. The data suggest that the above strain of the novel coronavirus can partially evade the neutralizing effect of antibodies (Abs) developed following the coronavirus infection or vaccination [30].

In addition, the B.1.1.523 strain should be mentioned; its frequency had significantly increased by May 2021. The fact that it has the E484K mutation shows that it (like the Beta strain) can be more resistant to Abs. Besides, this variant has alterations in the S protein – the S494P substitution and deletion of 3 amino acids. On the other hand, its proportion also sharply decreased in June 2021, when the Delta variant became the dominant variant. At the same time, we have not detected any cases of infection caused by the Gamma (Brazilian) strain.

Fig. 2 shows changes in the frequency of virus variants with N501Y or E484K mutations and not assigned to any of the specified strains due to the absence of other relevant changes in the genome.

Рис. 2. Представленность различных вариантов вируса SARS-CoV-2 с февраля по июнь 2021 г. на территории Москвы и Московской области.
Fig. 2. Representation of various variants of SARS-CoV-2 from February to June 2021 in Moscow and the Moscow Region.

Charts in Fig. 3 show changes in the prevalence rate for some mutations, which were previously identified as mutations altering the virus properties and causing increased transmissibility or evasion of the protective effect of Abs. For example, in February 2021, almost 15% of all isolates had the E484K mutation, though the Beta strain carrying this substitution had not been detected in Russia by that time. This fact proves that although such alterations in the genome can give certain advantages to the pathogen, their presence not always results in the widespread occurrence of the pathogen, if there are no other significant mutations, the combinations of which are frequently unclear. The subsequent months were characterized by an increase in the prevalence of several mutations, including E484K, N501Y, and S494P. In April 2021 when the Beta strain was detected in Russia, there was also an increase in the frequency of the K417N mutation, which is also present in the Delta Plus variant that was first detected in the Russian Federation only at the end of June 2021. Finally, L452R and T478K mutations belonging to the Delta strain occurred in May 2021 and are currently present in most of the SARS-CoV-2 genomes.

Рис. 3. Частота отдельных мутаций вируса SARS-CoV-2 в разные месяцы 2021 г. на территории Москвы и Московской области.
Fig. 3. Frequency of individual SARS-CoV-2 mutations in different months of 2021 in Moscow and the Moscow Region.

The obtained data show that by using a small primer panel for amplification of genomic fragments of the novel coronavirus and through the subsequent targeted sequencing, we can detect nearly all known alterations in the coronavirus S protein gene and identify strains of the pathogen, making it possible to monitor their frequency (prevalence) during specific periods. At the same time, it should be remembered that, by no means always, some mutations can result in emergence of a more contagious and epidemiologically dangerous variant of the virus; only in their combination, mutation changes can give new properties to the pathogen, sometimes causing its widespread occurrence.

Discussion

In this methodological work, we discussed the possibility to detect a number of SARS-CoV-2 strains, including Alpha (British, B.1.1.7), Beta (South-African, B.1.351), Gamma (Brazilian, P.1), Delta (Indian, B.1.617.2) variants, by using targeted high-throughput next-generation sequencing. For the above purpose, we designed a primer panel (Table) intended for efficient targeted amplification of genomic fragments of the novel coronavirus. The amplified regions include a large number of known significant mutations altering the properties of the virus, thus making it possible to decrease substantially the sequencing costs and to increase the number of samples under study. The latter advantage is highly important when significant changes in the S protein gene must be detected to identify the virus isolates belonging to different strains. To show how the primer panel is used, we studied 579 random SARS-CoV-2 samples collected from patients in Moscow and Moscow Region in February–June 2021. Note that most of the significant changes in the S protein gene are located in its small fragments primarily encoding amino acids found on the surface (Fig. 4). Our study also demonstrates the importance of promotion of population, genomic and epidemiological studies aimed at detection, prevalence tracking and monitoring of new variants of viral pathogens.

Рис. 4. Структурная модель S-белка, полученная с использованием криоэлектронной микроскопии (PDB ID: 7CAB).
а) – вид сбоку, б) – вид сверху.
Примечание. Числами от 417 до 501 обозначены аминокислотные остатки в соответствующих позициях. Регионы, вошедшие в праймерную панель, подсвечены синим цветом и показаны в виде небольших сфер. Значимые мутации (K417T, L452R, T478K, E484K, S494P, N501Y) показаны красным цветом как сферы большего диаметра и указаны на рисунке (б).
Fig. 4. Structural model of S-protein obtained using cryo-electron microscopy (PDB ID: 7CAB). View from the side (a) and from above (b).
Note. The numbers from 417 to 501 indicate the amino acid residues in the corresponding positions. The regions included in the primer panel are highlighted in blue and shown as small spheres. Significant mutations (K417T, L452R, T478K, E484K, S494P, N501Y) are shown in red as larger spheres and are indicated in figure (b).

We have shown a rapid change in the proportion of different genetic variants of the COVID-19 pathogen within the specified period, including the occurrence and swift spread of the Delta strain in Moscow and Moscow Region during May–June 2021; the above strain is partially responsible for the new wave of disease cases in Moscow this summer. In the meantime, the population’s inobservance of social distancing and personal protection measures as well as a low vaccination level are important factors boosting the spread of infection. Thus, the conducted study also demonstrates the importance of promotion of population, genomic and epidemiological studies aimed at detection, prevalence tracking and monitoring of the new SARS-CoV-2 variants and variants of other viral pathogens characterized by high variability.

Although we should assume that the new significant genomic variants may affect other important fragments of the viral genetic material, the using of a panel with few primers provides a substantial reduction in expenses on detection of currently circulating strains, while the simple design makes it possible to change promptly the structure of oligonucleotides, bringing in regular updates on genomes. It should be noted that targeted sequencing cannot completely replace whole genome one, which is essential for detecting all genomic alterations and a detailed phylogenetic analysis. For a detailed bioinformation analysis, specialists can use the recently built VGARus coronavirus genome database that as of the end of June 2021 had ~15,000 sequences, including ~8,000 complete genomes obtained through sequencing of isolates from different regions of Russia.

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About the authors

N. I. Borisova

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Author for correspondence.
Email: fake@neicon.ru
ORCID iD: 0000-0002-9672-0648

Moscow, 111123, Russia

Россия

I. A. Kotov

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor); FSAEI HE «Moscow Institute of Physics and Technology (National Research University)»

Email: fake@neicon.ru
ORCID iD: 0000-0003-2416-5689

Moscow, 111123, Russia

Moscow, 111123, Russia

Россия

A. A. Kolesnikov

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: fake@neicon.ru
ORCID iD: 0000-0003-3480-953X

Moscow, 111123, Russia

Россия

V. V. Kaptelova

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: fake@neicon.ru
ORCID iD: 0000-0003-0952-0830

Moscow, 111123, Russia

Россия

A. S. Speranskaya

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: fake@neicon.ru
ORCID iD: 0000-0001-6326-1249

Moscow, 111123, Russia

Россия

L. Yu. Kondrasheva

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: fake@neicon.ru
ORCID iD: 0000-0002-0147-4262

Moscow, 111123, Russia

Россия

E. V. Tivanova

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: fake@neicon.ru
ORCID iD: 0000-0003-1286-2612

Moscow, 111123, Russia

Россия

K. F. Khafizov

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: kkhafizov@gmail.com
ORCID iD: 0000-0001-5524-0296

Kamil F. Khafizov, Ph.D. (Biol.), Head of the Scientific Group for the Development of New Diagnostic Methods, Department of Molecular Diagnostics and Epidemiology

Moscow, 111123, Russia

Россия

V. G. Akimkin

FSBI «Central Research Institute for Epidemiology» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: fake@neicon.ru
ORCID iD: 0000-0003-4228-9044

Moscow, 111123, Russia

Россия

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Copyright (c) 2021 Borisova N.I., Kotov I.A., Kolesnikov A.A., Kaptelova V.V., Speranskaya A.S., Kondrasheva L.Y., Tivanova E.V., Khafizov K.F., Akimkin V.G.

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