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Fulltext Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa

Wong CF, Rahman RNZRA, Basri M, Salleh AB

Iran J Biotechnol, 2017;15(3):194-200.
PMID: 29845069 DOI: 10.15171/ijb.1524

Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.

Matched MeSH terms: Genetic Variation
Analysis of fliC variation among clinical isolates of Burkholderia cepacia

Winstanley C, Hales BA, Morgan JAW, Gallagher MJ, Puthucheary SD, CISSé MF, et al.

J Med Microbiol, 1999 Jul;48(7):657-662.
PMID: 10403416 DOI: 10.1099/00222615-48-7-657

PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type 11 flagellins were present. Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal. Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence. When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion. The variation in fliC sequences of B. cepacia was far greater than that with B. pseudomallei, supporting the view that 'B. cepacia', as currently defined, may comprise several different genomic species.

Matched MeSH terms: Genetic Variation/genetics*
Fulltext High genetic diversity of Enterococcus faecium and Enterococcus faecalis clinical isolates by pulsed-field gel electrophoresis and multilocus sequence typing from a hospital in Malaysia

Weng PL, Ramli R, Shamsudin MN, Cheah YK, Hamat RA

Biomed Res Int, 2013;2013:938937.
PMID: 23819125 DOI: 10.1155/2013/938937

Little is known on the genetic relatedness and potential dissemination of particular enterococcal clones in Malaysia. We studied the antibiotic susceptibility profiles of Enterococcus faecium and Enterococcus faecalis and subjected them to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). E. faecium and E. faecalis displayed 27 and 30 pulsotypes, respectively, and 10 representative E. faecium and E. faecalis isolates (five each) yielded few different sequence types (STs): ST17 (2 isolates), ST78, ST203, and ST601 for E. faecium, and ST6, ST16, ST28, ST179, and ST399 for E. faecalis. Resistance to tazobactam-piperacillin and ampicillin amongst E. faecium isolates was highly observed as compared to E. faecalis isolates. All of the isolates were sensitive to vancomycin and teicoplanin. The presence of epidemic and nosocomial strains of selected E. faecium STs: 17, 78, and 203 and E. faecalis ST6 as well as high rates of resistance to multiple antibiotics amongst E. faecium isolates is of a particular concern.

Matched MeSH terms: Genetic Variation*
Prediction of breast cancer risk based on common genetic variants in women of East Asian ancestry

Wen W, Shu XO, Guo X, Cai Q, Long J, Bolla MK, et al.

Breast Cancer Res, 2016 12 08;18(1):124.
PMID: 27931260

BACKGROUND: Approximately 100 common breast cancer susceptibility alleles have been identified in genome-wide association studies (GWAS). The utility of these variants in breast cancer risk prediction models has not been evaluated adequately in women of Asian ancestry.
METHODS: We evaluated 88 breast cancer risk variants that were identified previously by GWAS in 11,760 cases and 11,612 controls of Asian ancestry. SNPs confirmed to be associated with breast cancer risk in Asian women were used to construct a polygenic risk score (PRS). The relative and absolute risks of breast cancer by the PRS percentiles were estimated based on the PRS distribution, and were used to stratify women into different levels of breast cancer risk.
RESULTS: We confirmed significant associations with breast cancer risk for SNPs in 44 of the 78 previously reported loci at P

Matched MeSH terms: Genetic Variation*
Pepidase B variants among the Semai, Temuan, Semelai, and Jakun groups of West Malaysian Orang Asli

Welch QB

Hum. Hered., 1973;23(5):482-6.
PMID: 4785876
Matched MeSH terms: Genetic Variation*
Adenosine deaminase polymorphism among the Semai, Temuan, Semelai, and Jakun groups of West Malaysian Orang Asli

Welch QB, Shu LC, Thangavelu S, Lie-Injo EL

Hum. Hered., 1978;28(1):62-5.
PMID: 618819

812 West Malaysian Orang Asli belonging to four ethnic groups were surveyed for adenosine deaminase (ADA; EC 3.5.4.4) using starch gel electrophoresis. Only the common ADA1 and ADA2 alleles were found, with the frequencies of the latter being 0.025, 0.103, 0.115 and 0.028 in the Semai, Semelai, Temuan, and Jakun groups, respectively. A new 'breeding genetic distance' was applied to these gene frequencies and the Semelai and Temuan were found to be more closely related to each other, and to have considerably more evolutionary flexibility on this scale of 'micro-evolution' than the other two groups. The Semai and Jakun were more similar to each other on the basis of these ADA gene frequencies.

Matched MeSH terms: Genetic Variation
Serum albumin variants in three Malaysian racial groups

Welch QB, Lie-Injo LE

Hum. Hered., 1972;22(5):503-7.
PMID: 4670071
Matched MeSH terms: Genetic Variation*
Adenylate kinase and malate dehydrogenase in four Malaysian racial groups

Welch QB, Luan Eng LI, Bolton JM

Humangenetik, 1971;14(1):61-3.
PMID: 5144903
Matched MeSH terms: Genetic Variation*
Genomic analysis of some Japanese isolates of Getah virus

Wekesa SN, Inoshima Y, Murakami K, Sentsui H

Vet Microbiol, 2001 Nov 08;83(2):137-46.
PMID: 11557154

Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.

Matched MeSH terms: Genetic Variation*
Fulltext Population structure of the blood clam (Tegillarca granosa) in China based on microsatellite markers

Wang YJ, Zeng QG, Xu LN

Genet. Mol. Res., 2013;12(2):892-900.
PMID: 23613236 DOI: 10.4238/2013.April.2.6

The blood clam, Tegillarca granosa, is widely cultivated in China. We isolated 6 microsatellite loci from T. granosa and used them to investigate genetic diversity and population structure of 5 widely distributed populations of blood clam collected from eastern and southeastern China. The allele number per locus varied from 4 to 9, and the polymorphism information content value was 0.301 to 0.830. The mean observed and expected heterozygosities varied from 0.304 to 0.460 and 0.556 to 0.621, respectively; the population from Yueqing had the smallest observed heterozygosity. In the neighbor-joining tree, Shandong, Fenghua and Yueqing populations clustered together, and there was geographic divergence between Shandong and Guangxi populations. Some microsatellite loci that were isolated from these mainland China samples were not found in blood clams collected from Malaysia.

Matched MeSH terms: Genetic Variation
Fulltext Origin and diversity of an underutilized fruit tree crop, cempedak (Artocarpus integer, Moraceae)

Wang MMH, Gardner EM, Chung RCK, Chew MY, Milan AR, Pereira JT, et al.

Am J Bot, 2018 05;105(5):898-914.
PMID: 29874392 DOI: 10.1002/ajb2.1094

PREMISE OF THE STUDY: Underutilized crops and their wild relatives are important resources for crop improvement and food security. Cempedak [Artocarpus integer (Thunb). Merr.] is a significant crop in Malaysia but underutilized elsewhere. Here we performed molecular characterization of cempedak and its putative wild relative bangkong (Artocarpus integer (Thunb). Merr. var. silvestris Corner) to address questions regarding the origin and diversity of cempedak.
METHODS: Using data from 12 microsatellite loci, we assessed the genetic diversity and genetic/geographic structure for 353 cempedak and 175 bangkong accessions from Malaysia and neighboring countries and employed clonal analysis to characterize cempedak cultivars. We conducted haplotype network analyses on the trnH-psbA region in a subset of these samples. We also analyzed key vegetative characters that reportedly differentiate cempedak and bangkong.
KEY RESULTS: We show that cempedak and bangkong are sister taxa and distinct genetically and morphologically, but the directionality of domestication origin is unclear. Genetic diversity was generally higher in bangkong than in cempedak. We found a distinct genetic cluster for cempedak from Borneo as compared to cempedak from Peninsular Malaysia. Finally, cempedak cultivars with the same names did not always share the same genetic fingerprint.
CONCLUSIONS: Cempedak origins are complex, with likely admixture and hybridization with bangkong, warranting further investigation. We provide a baseline of genetic diversity of cempedak and bangkong in Malaysia and found that germplasm collections in Malaysia represent diverse coverage of the four cempedak genetic clusters detected.

Matched MeSH terms: Genetic Variation*
Phylogeography of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) on the host of mottled spinefoot (Siganus fuscescens) in the South China Sea, inferred from mitochondrial COI and ND2 genes

Wang M, Yan S, Brown CL, Shaharom-Harrison F, Shi SF, Yang TB

Mitochondrial DNA A DNA Mapp Seq Anal, 2016 11;27(6):3865-3875.
PMID: 25319302

To examine the phylogeographical pattern of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) in the South China Sea, fragments of mitochondrial cytochrome c oxidase subunit I and NADH dehydrogenase subunit 2 genes were obtained for 220 individuals collected from 8 localities along the southeast coast of China and 1 locality in Terengganu, Malaysia. Based on these two genes, two and three distinct clades with geographic signals were revealed on the phylogenetic trees respectively. The divergence between these clades was estimated to occur in the late Pleistocene. Analysis of molecular variance and pairwise FSTsuggested a high rate of gene flow among individuals sampled from the Chinese coast, but with obvious genetic differentiation from the Malaysian population. Mismatch distribution and neutrality tests indicated that the T. nebulosi population experienced expansion in Pleistocene low sea level periods. Vicariance was considered to account for the genetic divergence between Chinese and Malaysian populations, while sea level fluctuations and mainland-island connections during glacial cycles were associated with the slight genetic divergence between the populations along the mainland coast of China and those off Sanya. On the contrary, oceanographic circulations and host migration could lead to genetic homogeneity of populations distributed along the mainland coast of China.

Matched MeSH terms: Genetic Variation*
Fulltext Genetic diversity and differentiation of the orange-spotted grouper (Epinephelus coioides) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

Wang L, Meng Z, Liu X, Zhang Y, Lin H

Int J Mol Sci, 2011;12(7):4378-94.
PMID: 21845084 DOI: 10.3390/ijms12074378

In the present study, we employed microsatellite DNA markers to analyze the genetic diversity and differentiation between and within cultured stocks and wild populations of the orange-spotted grouper originating from the South China Sea and Southeast Asia. Compared to wild populations, genetic changes including reduced genetic diversity and significant differentiation have taken place in cultured grouper stocks, as shown by allele richness and heterozygosity studies, pairwise F(st), structure, molecular variance analysis, as well as multidimensional scaling analysis. Although two geographically adjacent orange-spotted grouper populations in China showed negligible genetic divergence, significant population differentiation was observed in wild grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia. However, the Mantel test rejected the isolation-by-distance model of genetic structure, which indicated the genetic differentiation among the populations could result from the co-effects of various factors, such as historical dispersal, local environment, ocean currents, river flows and island blocks. Our results demonstrated that microsatellite markers could be suitable not only for genetic monitoring cultured stocks but also for revealing the population structuring of wild orange-spotted grouper populations. Meanwhile, our study provided important information for breeding programs, management of cultured stocks and conservation of wild populations of the orange-spotted grouper.

Matched MeSH terms: Genetic Variation*
Fulltext Nine different glucose-6-phosphate dehydrogenase (G6PD) variants in a Malaysian population with Malay, Chinese, Indian and Orang Asli (aboriginal Malaysian) backgrounds

Wang J, Luo E, Hirai M, Arai M, Abdul-Manan E, Mohamed-Isa Z, et al.

Acta Med. Okayama, 2008 Oct;62(5):327-32.
PMID: 18985093

The Malaysian people consist of several ethnic groups including the Malay, the Chinese, the Indian and the Orang Asli (aboriginal Malaysians). We collected blood samples from outpatients of 2 hospitals in the State of Selangor and identified 27 glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects among these ethnic groups. In the Malay, G6PD Viangchan (871GA, 1311CT, IVS11 nt93TC) and G6PD Mahidol (487GA) types, which are common in Cambodia and Myanmar, respectively, were detected. The Malay also had both subtypes of G6PD Mediterranean:the Mediterranean subtype (563CT, 1311CT, IVS11 nt93TC) and the Indo-Pakistan subtype (563CT, 1311C, IVS11 nt93T). In Malaysians of Chinese background, G6PD Kaiping (1388GA), G6PD Canton (1376GT) and G6PD Gaohe (95AG), which are common in China, were detected. Indian Malaysians possessed G6PD Mediterranean (Indo-Pakistan subtype) and G6PD Namoru (208TC), a few cases of which had been reported in Vanuatu and many in India. Our findings indicate that G6PD Namoru occurs in India and flows to Malaysia up to Vanuatu. We also discovered 5 G6PD-deficient cases with 2 nucleotide substitutions of 1311CT and IVS11 nt93TC, but without amino-acid substitution in the G6PD molecule. These results indicate that the Malaysian people have incorporated many ancestors in terms of G6PD variants.
Study site: Kajang District Hospital and the Hospital Orang Asli Gombak, Selangor, Malaysia

Matched MeSH terms: Genetic Variation/genetics*
Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA

Walton C, Somboon P, O'Loughlin SM, Zhang S, Harbach RE, Linton YM, et al.

Infect Genet Evol, 2007 Jan;7(1):93-102.
PMID: 16782411

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.

Matched MeSH terms: Genetic Variation*
The isolation and characterization of microsatellites from Anopheles dirus mosquitoes

Walton C, Chang MS, Handley JM, Harbach RE, Collins FH, Baimai V, et al.

Mol Ecol, 2000 Oct;9(10):1665-7.
PMID: 11050564
Matched MeSH terms: Genetic Variation
Genetic variability of classical swine fever virus

Vilcek S, Stadejek T, Ballagi-Pordány A, Lowings JP, Paton DJ, Belák S

Virus Res, 1996 Aug;43(2):137-47.
PMID: 8864203

The genetic variability of classical swine fever virus was studied by comparative nucleotide sequence analysis of 76 virus isolates, collected during a half century from three continents. Parts of the E2 (gp55) and the polymerase gene coding regions of the viral genome were amplified by RT-PCR and DNA fragments of 254 and 207 bp, respectively, were sequenced. The comparative sequence analysis of the E2 region revealed two main phylogenetic groups of CSFV, indicating that the virus apparently evolved from two ancestor nodes. Group I (represented by Brescia strain) consisted of old and recent American and Asian viruses, as well as old English isolates from the 1950s. This group was subdivided into three subgroups, termed I.A-I.C. Group II (represented by Alfort strain) consisted of relatively recent isolates from Europe, together with strain Osaka, which was isolated in Japan from a pig of European origin. Based on genetic distances the group was divided into subgroups II.A and II.B. Malaysian isolates were branched into both groups, indicating multiple origins for contemporaneous outbreaks in that country. All ten vaccine strains tested were branched in group I, implying a common ancestor. The Japanese Kanagawa strain, isolated in 1974, and the British Congenital Tremor strain from 1964 were the most distinct variants of CSFV in our collection. The comparison of the nucleotide sequences of the polymerase coding region of 32 European strains distinguished subgroups II.A and II.B which were similar to the corresponding subgroups of the E2 phylogenetic tree. Thus, the results revealed that the E2 region and the polymerase coding regions seem to be appropriate for the grouping of CSFV isolates from all over the world, distinguishing two major groups of the virus. The reliability of these regions for phylogenetic analysis is indicated by the similarity of the results obtained from the two separate parts of the CSFV genome.

Matched MeSH terms: Genetic Variation*
Fulltext Phylogeography of the termite Macrotermes gilvus and insight into ancient dispersal corridors in Pleistocene Southeast Asia

Veera Singham G, Othman AS, Lee CY

PLoS One, 2017;12(11):e0186690.
PMID: 29186140 DOI: 10.1371/journal.pone.0186690

Dispersal of soil-dwelling organisms via the repeatedly exposed Sunda shelf through much of the Pleistocene in Southeast Asia has not been studied extensively, especially for invertebrates. Here we investigated the phylogeography of an endemic termite species, Macrotermes gilvus (Hagen), to elucidate the spatiotemporal dynamics of dispersal routes of terrestrial fauna in Pleistocene Southeast Asia. We sampled 213 termite colonies from 66 localities throughout the region. Independently inherited microsatellites and mtDNA markers were used to infer the phylogeographic framework of M. gilvus. Discrete phylogeographic analysis and molecular dating based on fossil calibration were used to infer the dynamics of M. gilvus dispersal in time and space across Southeast Asia. We found that the termite dispersal events were consistently dated within the Pleistocene time frame. The dispersal pattern was multidirectional, radiating eastwards and southwards out of Indochina, which was identified as the origin for dispersal events. We found no direct dispersal events between Sumatra and Borneo despite the presence of a terrestrial connection between them during the Pleistocene. Instead, central Java served as an important link allowing termite colonies to be established in Borneo and Sumatra. Our findings support the hypothesis of a north-south dispersal corridor in Southeast Asia and suggest the presence of alternative dispersal routes across Sundaland during the Pleistocene. For the first time, we also propose that a west-east dispersal through over-water rafting likely occurred across the Pleistocene South China Sea. We found at least two independent entry routes for terrestrial species to infiltrate Sumatra and Borneo at different times.

Matched MeSH terms: Genetic Variation
Fulltext Myrteae phylogeny, calibration, biogeography and diversification patterns: Increased understanding in the most species rich tribe of Myrtaceae

Vasconcelos TNC, Proença CEB, Ahmad B, Aguilar DS, Aguilar R, Amorim BS, et al.

Mol Phylogenet Evol, 2017 04;109:113-137.
PMID: 28069533 DOI: 10.1016/j.ympev.2017.01.002

Myrteae (c. 2500 species; 51 genera) is the largest tribe of Myrtaceae and an ecologically important groups of angiosperms in the Neotropics. Systematic relationships in Myrteae are complex, hindering conservation initiatives and jeopardizing evolutionary modelling. A well-supported and robust phylogenetic hypothesis was here targeted towards a comprehensive understanding of the relationships within the tribe. The resultant topology was used as a base for key evolutionary analyses such as age estimation, historical biogeography and diversification rate patterns. One nuclear (ITS) and seven chloroplast (psbA-trnH, matK, ndhF, trnl-trnF, trnQ-rps16, rpl16 and rpl32-trnL) DNA regions for 115 taxa representing 46 out of the 51 genera in the tribe were accessed and analysed using maximum likelihood and Bayesian inference tools for phylogenetic reconstruction. Dates of diversification events were estimated and contrasted using two distinct fossil sets (macro and pollen) in BEAST. The subsequent dated phylogenies were compared and analysed for biogeographical patterns using BioGeoBEARS and diversification rates using BAMM. Myrteae phylogeny presents strong statistical support for three major clades within the tribe: Australasian group, Myrtus group and Main Neotropical Lineage. Dating results from calibration using macrofossil are an average of 20 million years older and show an early Paleocene origin of Myrteae, against a mid-Eocene one from the pollen fossil calibration. Biogeographic analysis shows the origin of Myrteae in Zealandia in both calibration approaches, followed by a widespread distribution throughout the still-linked Gondwana continents and diversification of Neotropical endemic lineages by later vicariance. Best configuration shift indicates three points of acceleration in diversification rates, all of them occurring in the Main Neotropical Lineage. Based on the reconstructed topology, several new taxonomic placements were recovered, including: the relative position of Myrtus communis, the placement of the Blepharocalyx group, the absence of generic endemism in the Caribbean, and the paraphyletism of the former Pimenta group. Distinct calibration approaches affect biogeography interpretation, increasing the number of necessary long distance dispersal events in the topology with older nodes. It is hypothesised that biological intrinsic factors such as modifications of embryo type and polyploidy might have played a role in accelerating shifts of diversification rates in Neotropical lineages. Future perspectives include formal subtribal classification, standardization of fossil calibration approaches and better links between diversification shifts and trait evolution.

Matched MeSH terms: Genetic Variation
Fulltext NGS Technology in Monitoring the Genetic Diversity of Cytomegalovirus Strains

Vankova OE, Brusnigina NF, Novikova NA

Sovrem Tekhnologii Med, 2023;15(2):41-46.
PMID: 37389021 DOI: 10.17691/stm2023.15.2.04

Modern molecular genetic methods, massive parallel sequencing in particular, allow for genotyping of various pathogens with the aim of their epidemiological marking and improvement of molecular epidemiological surveillance of actual infections, including cytomegalovirus infection. The aim of the study is to evaluate the next-generation sequencing (NGS) technology for genotyping clinical isolates of cytomegalovirus (CMV).
MATERIALS AND METHODS: The object of the study were samples of biological substrates (leukocyte mass, saliva, urine) taken from patients who underwent liver and kidney transplantation. Detection of CMV DNA was carried out by a real-time PCR using commercial diagnostic AmpliSense CMV-FL test systems (Central Research Institute for Epidemiology, Moscow, Russia). DNA extraction was performed using DNA-sorb AM and DNA-sorb V kits (Central Research Institute for Epidemiology) in accordance with manufacturer's manual. The quality of the prepared DNA library for sequencing was assessed by means of the QIAxcel Advanced System capillary gel electrophoresis system (QIAGEN, Germany). Alignment and assembly of nucleotide sequences were carried out using CLC Genomics Workbench 5.5 software (CLC bio, USA). The sequencing results were analyzed using BLAST of NCBI server.
RESULTS: CMV DNA samples were selected for genotyping. The two variable genes, UL55(gB) and UL73(gN), were used for CMV genotype determination, which was performed using NGS technology MiSeq sequencer (Illumina, USA). Based on the exploratory studies and analysis of literature sources, primers for genotyping on the UL55(gB) and UL73(gN) genes have been selected and the optimal conditions for the PCR reaction have been defined. The results of sequencing the UL55(gB) and UL73(gN) gene fragments of CMV clinical isolates from recipients of solid organs made it possible to determine the virus genotypes, among which gB2, gN4c, and gN4b were dominant. In some cases, association of two and three CMV genotypes has been revealed.
CONCLUSION: The application of the NGS technology for genotyping cytomegalovirus strains can become one of the main methods of CMV infection molecular epidemiology, as it allows for obtaining reliable results with a significant reduction in research time.

Matched MeSH terms: Genetic Variation/genetics

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