Defense of the dissertation of Kamalova Dinara for the degree of Doctor of Philosophy (PhD) in the specialty «8D05107 - Biology»
L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Kamalova Dinara on the topic «Study of the genetic diversity of the SARS-CoV-2 virus and аssociated viral infections (enteroviruses and influenza A (H1N1) Virus) in Kazakhstan» to the educational program «8D05107 – Biology».
The dissertation was carried out at the «General Biology and Genomics education department» of L.N. Gumilyov Eurasian National University.
The language of defense is russian
Official reviewers:
Almagul Rakhimberliyevna Kushugulova - Doctor of Medical Sciences, Head of the Microbiome Laboratory, Center for Life Sciences, Private Institution “National Laboratory Astana”, Nazarbayev University (Astana, Kazakhstan).
Bayan Rysbekovna Turdalina - PhD, Professor, Pediatrician of the First Qualification Category, Associate Professor of the Department of Pediatric Infectious Diseases (Astana, Kazakhstan).
Temporary members of the Dissertation Council:
Kairat Kazybayevich Tabynov - PhD, associate professor, Kazakh national agrarian research university (Almaty, Kazakhstan).
Gulnur Sagindykovna Zhunusova - PhD, associate professor, leading researcher, Institute of genetics and physiology, committee of Science (Almaty, Kazakhstan).
Olesya Viktorovna Ohlopkova - candidate of biological sciences (PhD equivalent), Senior researcher, molecular virology laboratory, Smorodintsev research institute of influenza (Saint Petersburg, Russia).
Scientific advisors:
Alexander Borisovich Shevtsov - candidate of biological sciences, associate professor head of the laboratory of applied genetics National center for biotechnology LLP Astana, Kazakhstan.
Maxim Leonidovich Fillipenko - doctor of biological sciences, associate professor. head of the laboratory of pharmacogenomics Institute of chemical biology and fundamental medicine Siberian branch of the russian academy of sciences. Novosibirsk, Russian Federation.
The defense will take place on April 10, 2026, at 11:00 PM in the Dissertation Council for the training direction «8D051 – Biological and related sciences» in the specialty «8D05107 – Biology» of L.N. Gumilyov Eurasian National University. The defense meeting is planned to be held online.
Link: https://us06web.zoom.us/j/84209566920?pwd=Vb3hqH81YkdlgFj3xsbkhAGLokaSLi.1
ID: 842 0956 6920
Code: 461018
Address: Astana, Kazhymuqana str., 13, building №3, room №333
Abstract (English): General characteristics of the dissertation research The dissertation is devoted to a comprehensive investigation of the genetic diversity of the SARS-CoV-2 virus and associated viral infections - specifically, enteroviruses and influenza A (H1N1) - in patients from Kazakhstan during 2021 -2023, employing whole-genome sequencing methods. Relevance of the Study COVID-19 is an acute respiratory disease caused by the coronavirus SARS-CoV-2, which since 2020 has led to a global pandemic affecting millions worldwide. Despite the reduction in the severity of clinical forms as a result of vaccination and the accumulation of population immunity, the continuing circulation of new genetic variants of the virus maintains a high epidemiological risk. SARS-CoV-2 exhibits a high mutation rate, leading to the emergence of variants with altered transmissibility, pathogenicity, and resistance to neutralizing antibodies. Under conditions of active SARS-CoV-2 circulation within the population, cases of co-infection with other airborne viruses have acquired increasing significance. Particular attention is given to influenza A (H1N1) and enteroviruses, which may intensify the severity of the clinical picture of COVID-19, complicate diagnosis, and influence disease outcomes. The concurrent course of multiple viral infections necessitates timely detection and molecular typing of all pathogens involved. The study of the genetic diversity of SARS-CoV-2 and the monitoring of associated viral infections—such as influenza A (H1N1) and enteroviruses—are essential for understanding epidemic processes, optimizing laboratory diagnostic methods, and adjusting clinical protocols. This is especially relevant for Kazakhstan, where systematic research based on whole-genome sequencing and multiplex virological analysis had previously been lacking. Conducting such studies enhances the healthcare system’s preparedness for the emergence of new pathogens and co-infectious disease forms. Object of the Study RNA samples obtained from nasopharyngeal swab specimens collected from patients with a confirmed diagnosis of COVID-19. Objective of the Study To study the genetic diversity of the SARS-CoV-2 virus and associated viral infections (enteroviruses and influenza A (H1N1)) in Kazakhstan using whole-genome sequencing. Objectives of the Study 1. Primer design for whole-genome sequencing of SARS-CoV-2, taking into account harmonized annealing temperatures, the absence of secondary structures, and high specificity to the reference sequence NC_045512.2 (Wuhan-Hu-1). 2. Development of a reverse transcription and multiplex amplification protocol for comprehensive amplification of the SARS-CoV-2 genome. 3. Collection of biological specimens from patients with suspected respiratory viral infections (including COVID-19, influenza, and enteroviral infections) to enable a comprehensive assessment of circulating pathogens and their genetic diversity. 4. Whole-genome sequencing of SARS-CoV-2 using the Illumina MiSeq platform. 5. Comprehensive analysis of whole-genome sequencing data of SARS-CoV-2 obtained from patient RNA samples, including the identification of associations between viral genetic variants and clinical disease severity. Additionally, assessment of the prevalence and genetic diversity of enteroviruses and influenza A(H1N1) virus among patients with COVID-19 using whole-genome sequencing approaches. Research methods. The study employed microbiological, molecular-genetic, and bioinformatic methods of analysis, including polymerase chain reaction (PCR), reverse transcription, amplification, sequencing on the Illumina MiSeq platform, and data analysis using the Pangolin algorithm together with phylogenetic approaches. These complementary methodologies ensured reliable identification, amplification, and interpretation of complete viral genomes and associated co-infecting agents. Scientific novelty of the research results: - Primers and laboratory protocols for whole-genome amplification of SARS-CoV-2 were developed and optimized, enabling the generation of high-quality genomic sequences with a sequencing depth of 3000–5000×. - Whole-genome sequencing of 879 SARS-CoV-2 samples was performed, and sequences were classified using the Pangolin lineage assignment system. A total of 34 viral lineages were identified, including BA.5, B.1.617.2, XBB.1.5, and others, allowing the assessment of the temporal dynamics of dominant variant replacement in Kazakhstan. - For the first time in Kazakhstan, whole-genome sequencing of 65 enterovirus genomes was conducted in patients with COVID-19, revealing the circulation of eight serotypes (CVA9, CVB3, CVB5, E6, E9, E11, E21, and E25) based on complete VP1 gene sequence analysis. - Complete genome data of the influenza A(H1N1)pdm09 virus (A/Kazakhstan/5757/2022), belonging to clade 6B.1A.5a.2, were generated, providing the first documented evidence of circulation of this variant in Kazakhstan. - It was demonstrated that the presence of co-infecting viruses (including enteroviruses, influenza A(H1N1), Epstein–Barr virus, and others) influences the clinical course of SARS-CoV-2 infection, increasing the likelihood of complicated and atypical manifestations, particularly among pediatric patients. Theoretical and practical significance of the study: 1. Primer design for whole-genome sequencing of SARS-CoV-2 was performed, resulting in the selection of 39 primer pairs that divided the viral genome into 39 overlapping amplicons with a minimum overlap of 100 base pairs. The obtained data were used to develop a universal amplification panel suitable for diagnostic applications and population-level monitoring of viral variants. 2. A reverse transcription and multiplex amplification protocol was developed. Based on the designed primers, PCR conditions were optimized, and a whole-genome amplification protocol for SARS-CoV-2 was established using three multiplex reactions and a one-step RT-PCR approach. The developed protocol is suitable for genomic surveillance and the identification of emerging genetic variants of respiratory viruses. 3. Within the framework of this study, a collection of RNA samples was established, comprising 994 characterized specimens obtained from patients representing all regions of the Republic of Kazakhstan, including a cohort of children under 16 years of age treated at the Infectious Diseases Hospital in Astana. In addition, as part of monitoring for co-circulating viral infections, biological samples were collected from patients with suspected co-infections caused by enteroviruses and influenza A(H1N1) virus to evaluate their potential association with the clinical course of COVID-19. Whole-genome sequencing of SARS-CoV-2 was performed using the Illumina MiSeq platform. Between 2021 and 2023, a total of 881 whole-genome sequences were generated, exceeding the initially planned volume of 300 samples and substantially expanding the national genomic database of SARS-CoV-2 circulating in Kazakhstan. 4. An analysis of whole-genome sequencing data obtained from patient RNA samples was performed to identify associations with disease severity, period of infection, and treatment outcomes depending on viral genotype. According to Pangolin lineage classification, 879 sequences were assigned to 34 distinct lineages, the majority of which belonged to the Alpha, Delta, and Omicron variants. The most frequently circulating lineages in Kazakhstan were BA.5 of the Omicron variant (12.3%) and B.1.167.122 of the Delta variant (11.2%), both of which were also widely prevalent globally during the COVID-19 pandemic. Other commonly detected lineages included XBB.1.5 (8.88%), XBB.1.9 (8.66%), BA.1 (7.97%), B.1.617.2 (6.95%), B.1.1 (5.69%), AY.16 (4.56%), BQ.1 (3.64%), XBB (3.64%), B.4.1 (2.85%), A.2 (2.28%), and B.1.1.529 (2.16%). Less frequently circulating lineages included CH.1.1 (1.37%), B.1.1.7 (1.14%), B.1.617.2 (0.57%), B. (0.34%), B.1.1.189 (0.23%), B.1.1.294 (0.23%), B.1.617.2.127 (0.23%), BA.4 (0.23%), AY.122 (0.11%), BA.5.8 (0.11%), B.1.1.10 (0.11%), B.1.1.174 (0.11%), B.1.1.177.53 (0.11%), B.1.617.2.121 (0.11%), BA.5.2 (0.11%), and BA.5.8 (0.11%). Clinical manifestations of SARS-CoV-2 infection were largely determined by the genetic variant of the virus. Infection with the Delta variant was characterized by the predominance of respiratory symptoms such as cough and fever, while the frequency of gastrointestinal manifestations was markedly reduced. In addition, sore throat was more frequently observed in Delta infections compared to cases caused by the original wild-type strain. In contrast, infection associated with the Omicron variant was characterized by milder clinical manifestations compared with the Delta variant, particularly due to a lower probability of lower respiratory tract involvement. The analysis also demonstrated that a subset of pediatric patients exhibited atypical clinical presentations of SARS-CoV-2 infection in the context of viral co-infections (including Epstein–Barr virus, varicella-zoster virus, and rotavirus). These combined infections were associated with dermatological manifestations and pronounced gastrointestinal symptoms, reflecting the modifying effect of additional viral agents on the clinical spectrum of SARS-CoV-2 infection. 5. As part of the study, whole-genome sequencing of 65 enterovirus genomes isolated from patients during the seasonal surge of viral infections in 2022 was performed. Eight predominant serotypes were identified (Coxsackievirus A9, Coxsackievirus B3, Coxsackievirus B5, Echovirus 6, Echovirus 9, Echovirus 11, Echovirus 21, and Echovirus 25), and the genetic diversity of enteroviruses circulating in Kazakhstan was characterized for the first time. Additionally, in the autumn of 2022, the complete genome of influenza A(H1N1)pdm09 virus (A/Kazakhstan/5757/2022), belonging to clade 6B.1A.5a.2 and representative of global circulation in 2022, was sequenced for the first time in Kazakhstan. The obtained data demonstrate that whole-genome sequencing can be effectively applied not only for SARS-CoV-2 surveillance but also for monitoring co-circulating viral infections, thereby expanding the practical significance of the study for epidemiological surveillance and molecular diagnostics. Main propositions submitted for defense - Primer design for whole-genome sequencing of SARS-CoV-2 was performed, resulting in the selection of 39 primer pairs that divided the viral genome into 39 overlapping amplicons with a minimum overlap of 100 base pairs. - A laboratory protocol for whole-genome amplification of SARS-CoV-2 was developed and optimized, including the selection of specific primer pairs, reverse transcription methodology, and multiplex amplification approaches. The protocol ensures the generation of high-quality sequencing libraries and can be adapted for the analysis of other respiratory viruses. - Whole-genome sequencing of 881 SARS-CoV-2 samples collected across the Republic of Kazakhstan between 2021 and 2023 was performed using the Illumina MiSeq platform, followed by comprehensive bioinformatic analysis, providing a representative overview of viral evolution within the population. - The genetic diversity of SARS-CoV-2 circulating in Kazakhstan was characterized, with the identification of 34 genetic lineages, including BA.5, B.1.617.2, XBB.1.5, and others, confirming active viral evolution and temporal replacement of dominant variants. - An association between SARS-CoV-2 viral genotype and clinical characteristics of SARS-CoV-2-associated infection was demonstrated, including the influence of co-infecting viral agents (enteroviruses, influenza A(H1N1), Epstein–Barr virus, rotavirus, and varicella-zoster virus), highlighting the importance of genotyping in the interpretation of molecular epidemiological findings. - For the first time in Kazakhstan, whole-genome sequencing of enteroviruses isolated from patients with COVID-19 and during the seasonal increase of viral infections was performed. Minimum spanning phylogenetic trees were constructed, and eight serotypes were identified (Coxsackievirus A9, Coxsackievirus B3, Coxsackievirus B5, Echovirus 6, Echovirus 9, Echovirus 11, Echovirus 21, and Echovirus 25), enabling characterization of the genetic structure of circulating strains and their potential association with clinical manifestations. - The complete genome of influenza A(H1N1)pdm09 virus (A/Kazakhstan/5757/2022) sequenced in Kazakhstan was described for the first time and assigned to clade 6B.1A.5a.2, supporting the importance of including influenza virus surveillance within routine molecular monitoring programs alongside SARS-CoV-2. Main results and conclusions 1. Primers were designed to enable amplification of the entire SARS-CoV-2 genome in fragments ranging from 1200 to 1600 base pairs, with a minimum overlap of 100 base pairs between adjacent amplicons. 2. A laboratory protocol for whole-genome amplification of SARS-CoV-2 was developed and optimized, including the selection of specific primer pairs, reverse transcription methods, and multiplex amplification strategies. The protocol ensures uniform coverage of the entire viral genome with a sequencing depth ranging from 3000 to 5000×. 3. Biological specimens were collected from patients with suspected respiratory viral infections (including COVID-19, influenza, and enteroviral infections) to enable a comprehensive assessment of circulating pathogens and their genetic diversity. Within the framework of the study, a collection of 994 characterized samples was established, representing all regions of the Republic of Kazakhstan, including a pediatric cohort under 16 years of age from the Infectious Diseases Hospital in Astana. In addition, biological samples were collected from patients with suspected co-infections caused by enteroviruses and influenza A(H1N1) virus to evaluate their potential association with the clinical course of COVID-19. 4. The whole-genome sequencing protocol was optimized, and 881 SARS-CoV-2 samples underwent whole-genome sequencing. Novel data on genetic changes in SARS-CoV-2 were generated, providing comprehensive genomic information on viral strains circulating in Kazakhstan between 2021 and 2023. 5. The genetic diversity of SARS-CoV-2 circulating in Kazakhstan during 2021–2023 was characterized, and the temporal dynamics of dominant genotypes were analyzed. For the first time in Kazakhstan, clinical cases of pediatric COVID-19 complicated by co-infecting viral agents (Epstein–Barr virus, varicella-zoster virus, and rotavirus) were described. In addition, molecular genetic analyses of enteroviruses and influenza A(H1N1) virus were performed. Relation of the dissertation to state programs The research was carried out under the grant-funded program of the Committee of Science of the Ministry of Education and Science of the Republic of Kazakhstan for 2021–2023: AP09260668 “Study of the Genetic Diversity of the SARS-CoV-2 Virus in Kazakhstan.” Approbation of the work The main results of the dissertation were presented at the following scientific events: – III International Scientific and Practical Conference “Modern Pharmacy: New Approaches in Education and Current Research,” dedicated to the 70th anniversary of Professor Tanagul Akhimbayevna Arystanova, Laureate of the State Prize of the Republic of Kazakhstan.Oral presentation: “Clinical Characteristics of the Omicron BA.5 Variant in Children in Kazakhstan (2022–2023)” (Astana, 2023). – International Symposium “One Health – A Look into the Future,” organized by the Masgut Aikimbayev National Scientific Center for Especially Dangerous Infections in cooperation with the German Society for International Cooperation (GIZ) and the Bundeswehr Institute of Microbiology (IMB).Abstract presented: “Development of a Protocol for Reverse Transcription and Multiplex Amplification of the SARS-CoV-2 Genome” (Almaty, October 27, 2022). Publications based on the dissertation 1. Kamalova D. et al. “Co-infection with COVID-19 and Genetic Diversity of SARS-CoV-2 in Children.” Eurasian Journal of Applied Biotechnology (2023, No. 3, pp. 15–22). 2. Kamalova D. et al. “Development of a Protocol for Whole-Genome Sequencing of the SARS-CoV-2 Virus.” Bulletin of KazNU. Biological Series (2022, Vol. 92, No. 3, pp. 101–108). 3. Methodical Guidelines: “Whole-Genome Sequencing of SARS-CoV-2 Causing COVID-19 Infection.” Zhurinov M., Abilmagzhanov A.Z., Rakhimov K.D., Talgatov E., Korotetskiy I.S., Kamalova D.K. – Almaty: D.V. Sokolovsky Institute of Fuel, Catalysis and Electrochemistry JSC, 2023 – 28 p. (MR 01-2023). 4. “Genetic Diversity of Human Enterovirus in Kazakhstan, 2022.” International Journal of Microbiology (2024, Vol. 2024, No. 1, Article 7796913) – indexed in Web of Science and Scopus. 5. “Complete Genome Sequence of a Human Influenza A Virus (H1N1) Detected in Kazakhstan in the Fall of 2022.” Microbiology Resource Announcements (2025, Vol. 14, No. 1, e01040-24) – indexed in Web of Science Core Collection and Scopus (Q4). 6. A reverse-transcription and multiplex-amplification protocol for the SARS-CoV-2 genome (within eight reactions) was developed. 7. A complete whole-genome amplification protocol for the SARS-CoV-2 virus was created. Volume and structure of the dissertation. The dissertation comprises 131 pages of typed text and includes a literature review, materials and methods, research results, conclusion, list of references, and 5 appendices. The reference list contains 274 sources by domestic and international authors. The work includes 7 tables and 18 figures.
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