طب انتظامی (Jan 2022)
Evolutionary evaluation of the dominant genetic haplotype of SARS-CoV-2 virus in Iran from June to November 2020
Abstract
Introduction ... [1-13]. Preliminary studies identified 66 haplotypes and subhaplotypes on SARS-CoV-2 virus genomic sequences in 13 H1-H13 haplotype groups [14, 15]. These groups have been identified and defined as the most common genotyped haplotypes in viruses spread in the early stages of the COVID-19 pandemic [14, 15]. ... [16-18]. According to the data obtained from the studies of Dr. Elahi et al. On many sequences registered from other countries in the GISAID database, it has been found that three haplotypes (H5, H2, H1) among these haplotypes show the highest frequency compared to the others in the early stages of the COVID-19 epidemic [14]. Aim (s) This study aimed to evaluate the evolution of the dominant genetic haplotypes of the SARS-CoV-2 virus in Iran from June to November 2020. Research Society, Place and Time Viral RNA samples from this cross-sectional study, whose COVID-19 infection was confirmed by PCR test, were collected from individuals who were referred to one of the police force diagnostic laboratories in Tehran, Iran from June to November 2020. Sampling Method and Number The sample size was estimated to be 300 people. Sampling was performed completely randomly, regardless of age, sex, or severity of the disease in infected individuals. In selecting the samples, it was also tried not to select people with a relative ratio who were likely to be infected with a type of virus. The samples were included in the study in such a way that first the PCR test of the subjects should be positive and also the (Cycle threshold) CT of these samples should be between 14 and 24. Used Devices & Materials As a pattern, RNA samples were used to perform cDNA synthesis reactions using Mastermix cDNA synthesis (BIO FACT, Korea; Cat no: BR441-096). In these reactions, all three reverse PCR primers related to each of the three mutations were used. After cDNA synthesis from RNA samples, the ARMS-PCR technique was used to determine the genotype of nucleotides located in genomic positions T<14408C in haplotype H1 and T<8782C in haplotype H2 and mutation C<28688 in haplotype H5. For the ARMS-PCR response for mutations of T<14408C, T<8782C, and C<28688T, six primer pairs were designed using Gene runner v6.5.52Beta software to amplify the target genomic regions in such a way that the amplified fragment was amplified in the region. No mutation occurs only in the presence of the unchanged sequence-specific primer, and amplification of the fragment of the mutant region occurs only in the presence of the mutant-specific primer (Table 1). Therefore, for each synthesized cDNA sample, two series of PCR reactions were performed using MasterMix PCR (amplicon; 180301-50 2X Master Mix Red-Mgcl2: 15mM) and primers related to each change. The first reaction was with primers related to the non-mutated region and the second reaction was performed with the primers related to the mutated region. In haplotype H1, the length of the amplified fragment corresponding to the mutated region was 216 bp and the length of the amplified fragment related to the non-mutated region was 470 bp. Therefore, in electrophoresis of PCR products, mutated and non-mutated samples were observed on agarose gel with 216 and 470 bp bands, respectively. Also, in the studied change, in the H2 haplotype, a band with a fragment mutation of 240 bp is synthesized and in the normal sample, no fragment mutation with a length of 462 bp is synthesized. In the H5 haplotype, the amplitude of the amplified fragment for mutated and non-mutated regions was 214 and 493 bp, respectively. As a result, 214 bp and 493 bp bands were observed by electrophoresis of PCR products on an agarose gel. Then, to verify the results of genotyping the target genomic loci, the genomic fragments of 20 samples were sequenced by the Sanger sequencing method. Sequences were analyzed using Sequencher.V5.4.6 software. The Wuhan-Hu-1 isolated genome sequence (NC_045512.2) was also used as the SARS-CoV-2 reference sequence using the NCBI database. Ethical Permissions This plan was approved by the Research Council of the Deputy of Health of the police force in 2020 with the code 990706/03/33 and all ethical considerations were observed. Finding by Text A total of 291 valid RNA samples were collected from 300 cases of genomic material extracted from patients, and the dispersion of the samples received during different months was recorded (Table 2). The age of the subjects was reported to be between 17 and 66 years, which included both males and females. After cDNA synthesis and ARMS-PCR reactions and electrophoresis of the obtained products, it was observed that a total of 122 samples with CSanger sequencing also confirmed these results (Figures 1 and 2 c). The frequency of carriers was also compared by sample collection months. In the first months of the study period (June), the H5 haplotype group was the first group to be observed in the Iranian patient population after the COVID-19 epidemic, but gradually due to H1 haplotype prevailed due to the dynamics of the virus genome and new changes in the genome; So that in the last months of the selected period (November), in our samples, haplotype H1 reached a frequency of nearly 100% (Figure 3). While examining the samples in the period of six months, respectively, from June to November, 11 samples (0-1-3-2-3-2) with two nucleotides were observed in one place, which means that the person infected with two viruses has genetic haplotypes. Was infected differently. At the beginning of the study period, when only the H5 haplotype was the dominant haplotype, no specimens with two different nucleotides were observed at the same site, but over time, both H1 and H5 viral haplotypes were observed in the population. 11 infected samples were observed that were infected with both H1 and H5 viral haplotypes (Figures 1 and 2b). H2 haplotype was also the first group to appear in a very short time in the early days of the COVID-19 epidemic in East Asia, including China. The study also tested this haplotype group, which was not found in any of the sequences, and as a result, it was predicted that the H2 haplotype group was most likely removed from the viral population. Therefore, no haplotype of H2 was observed in this project. The results of this study also showed that all 291 samples that were PCRed were in two groups of haplotypes H1 and H5. Main Comparison to the Similar Studies The first virus sequence from Iran was uploaded on April 4 in GISAID (EPI_ISL_424349). In the same study, Eden et al. observed three major mutations in G1397A, T28688C, and G29742T in the genomes of patients with a history of travel to Iran, representing a distinct haplotype group of specific viral variants present in Iran at the time [19] and indicating the first. The sequence is available to the Iranian population with the H5 strain. As of October 23, 2020, there were only eight complete genomes in GISAID that were not sufficient to detect the virus in the country, and in the only epidemiological study of the outbreak in Iran, they used a genomic sequence specified from travelers to Iran. After some time, Najmabadi et al. In their study performed 50 samples of the virus genome in Iran incomplete genomic sequencing. The results of Najmabadi et al.'s research show that the primary viruses infecting the Iranian population are in two phylogenetic branches B4 and B1 [20]. Comparing the results obtained from the Najmabadi study and the present study, it was found that branch B1 was the same haplotype group H5 and branch B4 was the same haplotype group H1 [21-23]. Genotyping approaches in this study will significantly reduce research costs by detecting the dominant strain of the population using the simple and inexpensive ARMS-PCR technique instead of the costly whole virus genome sequencing technique. To the best of our knowledge, no such project has been reported in Iran before. ... [24, 25]. These mutations can be interpreted as meaning that the virus can make changes to its genome depending on the conditions in which it is located, to increase its proliferation rate or its binding power by altering the structure of the tentacles to cell surface receptors. Limitations Due to temperature sensitivity, the ARMS method requires great care in setting up and obtaining the correct result. To confirm the correct operation of the method, it must be randomly controlled by the sequencing method, which was one of the limitations of this study. Suggestions Due to the frequent changes in the virus genome and the possibility of creating new haplotypes with different phenotypic effects, it is suggested that the changes be examined periodically until the complete termination of the disease. Conclusions Estimates indicate that the predominant population of viruses infecting Iranians at the beginning of the epidemic had the H5 haplotype. Following the significant expansion of the H1 haplotype group worldwide and over time, this group replaced H5 as the dominant haplotype. H1-type sequences have significant changes in the RdRp and S genes, which are involved in virus replication and binding to the host cell, respectively. Clinical & Practical Tips in Police Medicine By identifying the dominant strain of the population in the target range at a much lower cost than complete genomic sequencing, a prelude to other epidemiological studies in the police community was provided. Acknowledgments We thank the medical staff of the police hospital, especially the genetic laboratory, the patients studied in the project, and other professors and people who helped us in this project. Conflict of Interest The authors state that there is no conflict of interest in the present study. Funding Sources The Deputy of Health police headquarter was the financial sponsor of this research.
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