The project plans to conduct genetic characterization by sequencing of SARS-CoV-2 variants that caused severe consequences in patients from Kazakhstan. Changes in the prevalence of important single nucleotide polymorphisms of the virus in the Kazakhstani population as the epidemic progresses will be investigated. Subgenomic RNA of the virus will be screened at different stages of disease progression in several patients. The project will also clone open reading frames of SARS-CoV-2 polypeptides, which are the most promising targets for diagnostic purposes and vaccine development. The cloned cDNAs will be expressed in bacteria and the synthesized recombinant proteins will be isolated and purified. The results obtained will serve as a basis for the development of domestic test systems based on PCR and ELISA for diagnosis, typing and prognosis of COVID-19.
The variability of the SARS-CoV-2 genome may soon negatively affect the efficacy of vaccines and diagnostics being developed for the virus. Therefore, it is necessary to continuously monitor which genetic variants of the virus are circulating in different human populations in order to maintain the disease control system at a high level.There are several approaches to assess the genetic variability of virus genomes.
The first approach is based on reading the entire viral genome and then analyzing the complete genomic libraries. This approach allows rapid identification of the most frequently occurring polymorphisms. Whole-genome sequencing is a highly expensive and complex method of analysis; recently, it is usually performed on next-generation sequencers (NGS) [19, 21].
The second approach involves partial sequencing of the most important regions of the viral genome. This approach saves significant resources and allows to focus only on the most informative parts of the genome. Researchers of SARS-CoV-2 variability usually choose the S protein gene (which is highly variable) as the main target: it is the S-protein of SARS-CoV-2 that produces the most viral neutralizing antibodies [23, 24], and substitutions in the main surface protein of coronaviruses can affect its binding by antibodies. Importantly, the most candidate subunit and vector recombinant vaccines are being developed based on the S protein and its individual regions [22]. In addition to the S protein, the structural proteins M and N are immunodominant in viruses: these three proteins contain the largest number of epitopes for MHCI and MHCII presentation [25-26]. The nucleocapsid protein N is the most immunodominant and the most conserved of these three proteins. In addition, it is not subject to glycosylation. These characteristics make it a convenient target for the development of ELISA-based test kits for the detection of SARS-CoV-2. The N protein gene is often chosen as a target for SARS-CoV-2 RNA detection by PCR [27-29] because of its conserved nature and the highest copy number compared to other coronavirus genes (due to the 3′-terminal dislocation of the N gene in gRNA, it is present in all sgRNAs of the virus) [30].
The third approach to genetic characterization of SARS-CoV-2 genomes involves the identification of specific, already established polymorphisms (more often single nucleotide polymorphisms, SNPs) in the human population using rapid, easy-to-analyze methods based on PCR or nucleic acid hybridization.
The present project plans to use all three of the above approaches for genetic characterization of SARS-CoV-2 genotypes circulating in Kazakhstan. Full genomic sequencing for a limited number of samples is planned to be performed using the MinION platform (OxfordNanoporeTechnologies). Partial (targeted) sequencing using the Sanger method is planned for samples isolated from patients with the most severe clinical manifestations, including fatalities. Point SNPs (such as 241C/T and 23403A/G) will be determined on a large number of PCR-positive samples. The novelty of the study lies, first of all, in the fact that samples obtained from Kazakhstanis will be analyzed.
The proposed project plans to analyze not only genetic but also subgenetic characteristics of the coronavirus. Since active viral replication and production of coronavirus sgRNA is mainly observed during the viremic stage, it has been suggested thatSARS-CoV-2 sgRNA can be used as a marker to establish the stage of coronavirus infection [31]. During COVID-19 outbreaks, hospitals are often overcrowded and residual amounts of coronavirus RNA can remain in the human body for a long time and give a positive response by PCR, even if the patient’s specific T- and B-cell immune response is active and virus multiplication is stopped, minimizing the chance of infecting other people. This type of biomarker could therefore be invaluable to health care workers involved in the control of new infection to differentiate highly infectious patients from recovering individuals with low infectious potential. At the same time, there are indications that the sgRNAs of the novel coronavirus may be associated with the cell membrane of cells, making them more stable than previously thought [32]. This project plans to test the validity of using different subgenomic RNAs as a biomarker to determine the stage of coronavirus infection to determine the feasibility of developing a domestic test system to detect SARS-CoV-2 sgRNAs. This study will also utilize in-house designed primers and assays to detect several different sgRNAs, rather than just one. The research conducted under the project will become a foundation for the development of various domestic PCR and ELISA-based test systems for the detection and/or prognosis of COVID-19.
to conduct population studies on SARS-CoV-2 genotypes circulating in Kazakhstan during the 2020 pandemic, analyze their subgenomic characteristics and obtain recombinant SARS-CoV-2 proteins to develop science-based approaches to the diagnosis of COVID-19.
The project will produce the following direct scientific results:
– DNA constructs containing genome fragments of domestic SARS-CoV-2 genotypes;
– expression lines of E. coli strains expressing target cDNA genes of SARS-CoV-2;
– purified recombinant SARS-CoV-2 proteins;
– new primers and fluorescently labeled probes for SARS-CoV-2 typing and detection of its subgenomic RNA.
The results obtained in the course of the project will help to determine which domestic test systems should be developed to improve the efficiency of diagnosis and prognosis of COVID-19. Data on the genotypes of the new coronavirus circulating at different stages of the epidemic in Kazakhstan will be obtained. The primers and assays developed during the project, as well as genetic constructs carrying sections of the viral genome, can be used to develop domestic RT-qPCR-based test systems for detection and typing of SARS-CoV-2, as well as for predicting the severity of the disease. The isolated recombinant proteins can be used to develop a domestic ELISA-based test system for the detection of antibodies to SARS-CoV-2.
Skiba Y.A., Project Manager, PhD in Biological Sciences (Molecular Biology). Hirsch Index: 6. ORCID: http://orcid.org/0000-0003-4895-1473. Scopus ID: 56677594900. WoSID: H-6528-2017.
Nizkorodova A.S., Candidate of Biological Sciences (molecular biology). Hirsch index: 1. ORCID: https://orcid.org/0000-0002-1597-7207. Scopus ID: 57215971184. WoSID: AAY-1646-2020.
Dmitrovsky A.M., Doctor of Medical Sciences, Professor, Head of Laboratory, Infectious Diseases Physician. Hirsch Index: 2. ORCID: https://orcid.org/0000-0003-4714-3079. ScopusID: 57204864464. WoSID: AAZ-2816-2020.
Ismagulova G.A. – Candidate of Biological Sciences (molecular biology). Hirsch Index: 1. ORCID: https://orcid.org/0000-0002-2735-4939. ScopusID: 6506396016. WoSID: AAO-3437-2020.
Berdygulova J.A., PhD- student (biotechnology), specialist in virology and molecular biology. Hirsch Index: 4. ORCID: https://orcid.org/0000-0003-0379-2472. Scopus ID: 23977664200. WoS ID: I-2943-2018.
Neupokoyeva A.S., Master of Biotechnology. Hirsch Index: 1. ORCID: http://orcid.org/0000-0001-7257-8037. ScopusID: 57217703182. WoSID: N-9341-2017.
Berezovsky D.V., M.Sc. (Medicine), doctor of hygienist-epidemiologist. Hirsch index: 1. ORCID: https://orcid.org/0000-0002-2830-1994. ScopusID: 57212528777. WoSID: AAY-6245-2020.
Naizabaeva D.A., Master of Biotechnology. ORCID: http://orcid.org/0000-0002-0606-4289. Scopus ID: 57218288692.WoS ID: AAY-5696-2020.
2021 year
Partial sequencing of the SARS-CoV-2 genome was performed with RNA isolated from samples of Kazakhstani patients with severe disease course, samples from pathologic material.
Primers were designed on the basis of analysis of nucleotide sequences of SARS-CoV-2 gene ORFin MEGA-X program and analysis of secondary structure of primers and duplexes in RNA-structure program; primers were synthesized and purified in the laboratory of organic synthesis of RSE “NCB” KN MES RK. Unique long amplifications including the whole ORF of the spike-in protein SARS-CoV-2 were obtained for single RT-PCR of positive RNA samples, and two amplifications completely overlapping this ORF were obtained using the primer pair CoV-Lead-F/RvS and primer pair FwS/Sfull_Kpn_R. After purification of the amplifications, Sanger sequencing reaction was performed using the synthesized primers.
Samples from patients with severe disease course, samples from pathological material, samples from COVID-19 re-infected and re-infected individuals with moderately severe disease course, and from vaccinated individuals who were infected with COVID 19 and exhibited severe disease symptoms were sequenced. For the twelve RNA-positive samples, it was possible to overlap the full ORF sequence of the surface glycoprotein (adhesion protein S) with the rids. In all eleven variants, the 23403A > G mutation (resulting in a D614G amino acid substitution in the S protein) was identified. In one sample, in addition to the 23403A > G substitution, an A/C nucleotide mix was detected at position 23208. The nucleotide sequence of the ORFS-protein of seven samples was no longer different from the original sequence of the “Wuhan” genetic variant. The sequences of three samples contained all substitutions characteristic of the British alpha strain (WHO classification) of SARS-CoV-2 (S: HV69-70del; S: Y144del; S: N501Y; S: A570D S: D614G; S: P681H; S: T716I; S: S982A; S: D1118H). One sample contained all of these mutations (characteristic of an alpha strain of the virus according to the WHO classification) except for the S:T716I substitution. The sequences of these twelve ORFof the spike-in protein are posted in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank): GenBank numbers OK354348 through OK354359.
2022 year
PCR-positive samples collected at different stages of the COVID-19 epidemic in Kazakhstan were screened to detect the most important single nucleotide polymorphisms (SNPs) of SARS-CoV-2.
– The design of primers and samples for detection of the most important mutations of SARS-CoV-2 by classical RT-PCR (detection of SNPs, deletions and insertions) and RT-qPCR (SNPs) was carried out. They were synthesized de novo and purified by HPLC.
– From April 01, 2020 to April 30, 2022, RT-PCR-positive total RNA preparations by COVID-19 obtained from patients were selected from April 01, 2020 to April 30, 2022.
– The first important mutation in the S gene region encoding the furin-recognizing site of the SARS-CoV-2 fusion protein was screened by RT-qPCR method.
– Mutations were screened by classical RT-qPCR to differentiate VOCs from each other and from variants not classified as VOCs.
Primers and probes were designed, synthesized and purified to detect the most important single nucleotide polymorphisms (SNPs) of SARS-CoV-2 by PCR.
– Nucleotide sequences of genetic variants that were approved by WHO as notifiable (VOCs), namely alpha (B.1.1.7), beta (B1.351), gamma (B.1.1.248), delta (B.1.617+) and omicron (B.1.1.529).
– Primers were designed, synthesized and purified to detect the most important mutations (SNPs, deletions and insertions) allowing differentiation of all five genetic variants of SARS-CoV-2 VOCs by classical PCR.
– We selected, synthesized and purified primers and probe for detection of the “23403A > G” mutation (S: D614G) by RT-qPCR (real-time PCR).
PCR-typing of viral RNA isolated from samples received at different stages of the epidemic in Kazakhstan was performed.
– Fifty RT-PCR positive samples at the initial stage of the pandemic (April-September 2020) were analyzed to detect the 23403A > G” mutation (S: D614G). Only one sample (from the earliest) was identified that did not contain the mutation.
– At different stages of the COVID-19 pandemic, a representative sample of samples (from two hundred patients) that gave a positive response by RT-qPCR with the lowest Ct value was compiled.
– The 200 PCR-positive samples were typed with respect to whether or not they belonged to one or another SARS-CoV-2 VOC. No gamma variant (B.1.1.248) was detected. Only one beta variant (B1.351) was identified. Thirty-seven samples of variant alpha (B.1.1.7), 25 samples of variant delta (B.1.617+) and 39 samples of variant omicron (B.1.1.529) were identified.
– An electronic database on SARS-CoV-2 genetic variants was created.
2023 year
– Based on literature data, we selected SARS-CoV-2 polypeptides that are the most promising targets for diagnosis and vaccine development: spike protein S, membrane protein M, nucleoprotein N, and structural protein E. The nucleotide sequences of the regions of the viral genome encoding these structural proteins of coronavirus were analyzed in the Vector NTI 11. 0 program, we analyzed the nucleotide sequences of the viral genome regions encoding these structural proteins of the coronavirus for the main notified genetic variants (alpha, beta, gamma, delta, omicron) and the original genetic variant. The sites with the smallest genetic differences between the major genetic variants of SARS-CoV-2 were selected, and all nucleotide sequences were analyzed for the presence of restriction sites. The primers for cloning of the above mentioned SARS-CoV-2 ORFs were designed, and the corresponding restriction sites were analyzed for cloning the amplifiers into the expression vector E. coli pET23c. All primers were synthesized and purified.
– In order to develop potential targets for ELISA kits, the ORFs of adherens junction surface glycoprotein S, membrane protein M, nucleoprotein N, and porin protein E were cloned. Only cDNA encoding the region of S protein with extramembrane localization was used to clone the ORFof S protein. PCR using a high-precision enzyme was performed. Purified amplifications were treated with restriction endonucleases: for cDNA-gene S – NheI/XhoI; for cDNA-gene N – NheI / SalI; for cDNA-gene M – NdeI/XhoI; for cDNA-gene E – NdeI / SalI. Amplifications purified after restriction were ligated into vector plasmid pET23c using NdeI / SalI or NheI/SalI restriction sites. The ligase site was transfected into competent cells of E.coli DH5 strain. From the grown DNA clones, plasmids were typed and isolated using chloroform method. DNA clones were tested by restriction analysis using XbaI/ Bpu1102I restriction endonucleases. The presence of insertions was also confirmed by PCR analysis using gene-specific primers. To verify the cloned nucleotide sequences, partial sequencing (only the cloned DNA section) of plasmids pET23c_SARS-CoV2-S, pET23c_SARS-CoV2-N, pET23c_SARS-CoV2-M, pET23c_SARS-CoV2-E was performed.
– For amplification of SARS-CoV-2 structural proteins ORFs, an RNA preparation from nasopharyngeal flush was used as a matrix, for which the entire structural region of the viral genome of the S lineage (B.1.1) had previously been read by Sanger DNA sequencing. Using high-precision Phusion polymerase and specific primers containing restriction sites for cloning, ORFamplifications of the S, M, N, and E structural proteins of SARS-CoV-2 were obtained. In the case of the spike protein S, not the full-length ORFof this protein was amplified, but its truncated region without the sites encoding the signal peptide, transmembrane and cytosolic segments of the spike protein.
– Amplifications of appropriate sizes were obtained (3626 bp for the S gene cDNA; 1283 bp for the N gene cDNA; 694 bp for the M gene cDNA; 252 bp for the E gene cDNA). After purification and treatment with appropriate restriction endonucleases, the amplifiers were cloned into the expression vector pET23c, which allows expression of cDNA regions cloned into it in bacterial cells. Cloning was performed so that six triplets encoding histidine were added to the target ORFfrom the 3′-end in the same frame as them. Plasmid maps of pET23c_SARS-CoV2-S, pET23c_SARS-CoV2-N, pET23c_SARS-CoV2-M, and pET23c_SARS-CoV2-E were constructed in the Vector NTI program. Sanger sequencing of cloned DNA regions was performed from the T7 promoter.
– Sequenced plasmids were produced in midi-prep quantities and isolated using GeneJET Plasmid Midiprep Kit (Thermo Fisher Sci.).
– After transformation of cells of E. coli Bl-21(DE3) expression strain with DNA constructs, ORFexpression of target proteins was performed. Cells carrying plasmid pET23c_SARS-CoV2-N practically did not grow at temperatures above 30 ºC. Before the addition of IPTG, they were allowed to grow at 28 ºC, and after the addition of lac operon activator – at 25 ºC. The bacterial cells transformed by plasmids pET23c_SARS-CoV2-E and pET23c_SARS-CoV2-M grew normally before the addition of IPTG, but after its addition to the nutrient medium the optical density of cell suspensions did not increase with time, which indicated that the viral proteins synthesized in them were toxic for bacteria. To produce M-6His and E-6His proteins, the optimization of cDNA expression conditions involved reducing the IPTG concentration from 0.5 mM to 0.1 mM and reducing the time of induction of cDNA expression from four hours to one. Cells transformed with pET23c_SARS-CoV2-S plasmid showed standard growth at temperatures above 30 ºC, and no problems with cDNA gene S expression were recorded.
– After the recombinant proteins were made in bacterial cells, their ability to convert to a soluble form was tested. Only recombinant protein N-6His was detected in the supernatant after lysis of bacteria and centrifugation of the lysate at 20000 g for 20 min.
– S-6His, M-6His, and E-6His proteins were consistently localized in the precipitate (i.e., formed insoluble inclusion bodies or associated with membranes) and were almost completely absent in the supernatant (data not shown). Addition of detergents (1% Tween-20, 1% Triton X-100 or 1% SDS) to the lysing solution did not solve the problem: some fraction of S-6His was transferred to the soluble fraction, but dimers and other aggregates were formed (data not presented). In the case of M-6His and E-6His proteins, the addition of detergents to the lysing solution had no effect on their solubilization.
– S-6His, M-6His, and E-6His proteins were purified by Ni-NTA agarose under denaturing conditions, and N-6His protein was purified under native conditions by affinity chromatography. The Ni-NTA agarose eluted proteins were purified by dialysis to get rid of the imidazole or denaturing agents they contained and to adjust their pH to 7.5. After dialysis, the proteins were concentrated. The synthesized proteins were analyzed by Western blotting. Proteins from 12.5% PAA gel were transferred to nitrocellulose membrane by semi-dry blotting. His-tag labeled proteins were detected using monoclonal antibodies to the 5-His sequence (first antibodies), and proteins were stained using chemiluminescent substrate.
– All four SARS-CoV-2 proteins (N, S, E, and M) were synthesized and purified. The purity of these proteins ranged from 10 to 25% of all proteins in the eluates. The total amounts of full-length misfolded and purified proteins (from 50 mL of the original cell suspension) were: 296.2 μg (S protein), 277.8 μg (N protein), 137.6 μg (M protein), and 128.2 μg (E protein).