Phylogenetic Analyses Suggest that Factors Other Than the Capsid Protein Play a Role in the Epidemic Potential of GII.2 Norovirus

ABSTRACT Norovirus is the leading cause of acute gastroenteritis worldwide. For over two decades, a single genotype (GII.4) has been responsible for most norovirus-associated cases. However, during the winter of 2014 to 2015, the GII.4 strains were displaced by a rarely detected genotype (GII.17) in several countries of the Asian continent. Moreover, during the winter of 2016 to 2017, the GII.2 strain reemerged as predominant in different countries worldwide. This reemerging GII.2 strain is a recombinant virus that presents a GII.P16 polymerase genotype. In this study, we investigated the evolutionary dynamics of GII.2 to determine the mechanism of this sudden emergence in the human population. The phylogenetic analyses indicated strong linear evolution of the VP1-encoding sequence, albeit with minor changes in the amino acid sequence over time. Without major genetic differences among the strains, a clustering based on the polymerase genotype was observed in the tree. This association did not affect the substitution rate of the VP1. Phylogenetic analyses of the polymerase region showed that reemerging GII.P16-GII.2 strains diverged into a new cluster, with a small number of amino acid substitutions detected on the surface of the associated polymerase. Thus, besides recombination or antigenic shift, point mutations in nonstructural proteins could also lead to novel properties with epidemic potential in different norovirus genotypes. IMPORTANCE Noroviruses are a major cause of gastroenteritis worldwide. Currently, there is no vaccine or specific antiviral available to treat norovirus disease. Multiple norovirus strains infect humans, but a single genotype (GII.4) has been regarded as the most important cause of viral gastroenteritis outbreaks worldwide. Its persistence and predominance have been explained by the continuous replacement of variants that present new antigenic properties on their capsid protein, thus evading the herd immunity acquired to the previous variants. Over the last three seasons, minor genotypes have displaced the GII.4 viruses as the predominant strains. One of these genotypes, GII.2, reemerged as predominant during 2016 to 2017. Here we show that factors such as minor changes in the polymerase may have driven the reemergence of GII.2 during the last season. A better understanding of norovirus diversity is important for the development of effective treatments against noroviruses.

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Norovirus Structure and Classification

10.5772/intechopen.98216 ◽

2021 ◽

Author(s):

Manisha Rani ◽

Sushma Rajyalakshmi ◽

Sunitha Pakalapaty ◽

Nagamani Kammilli

Keyword(s):

Capsid Protein ◽

Diarrheal Disease ◽

Herd Immunity ◽

Significant Proportion ◽

Point Mutations ◽

Human Infection ◽

Sequence Difference ◽

Structural Protein ◽

Single Genotype ◽

Capsid Protein Vp1

Norovirus are a major cause of acute gastroenteritis worldwide. Diarrheal disease is now the fourth common cause of mortality children under the age of 5 years but remain the 2nd most cause of morbidity. NoV are associated with 18% diarrheal diseases worldwide where rotavirus vaccinations has been successfully introduced. NoV has become major cause of gastroenteritis in children. NoV belong to family caliciviridae. They are non-enveloped, single stranded positive sense RNA Viruses. The genome consists of 3 Open reading frames, ORF-1 codes for non-structural protein, ORF-2 codes for major capsid protein VP1 and ORF-3 for minor capsid protein VP2. Based on sequence difference of the capsid gene (VP1), NoV have been classified in to seven genogroup GI-GVII with over 30 genotypes. Genogroups I, II, IV are associated with human infection. Despite this extensive diversity a single genotype GII.4 has been alone to be the more prevalent. Basic epidemiological disease burden data are generated from developing countries. NoV are considered fast evolving viruses and present an extensive diversity that is driven by acquisition of point mutations and recombinations. Immunity is strain or genotype specific with little or no protection conferred across genogroups. Majority of outbreaks and sporadic norovirus cases worldwide are associated with a single genotype, GII.4 which was responsible for 62% of reported NoV outbreaks in 5 continents from 2001 to 2007. GII.4 variants have been reported as major cause of global gastroenteritis pandemics starting in 1995 frequent emergence of novel GII.4 variants is known to be due to rapid evolution and antigenic variation in response to herd immunity. Novel GII.4 variants appear almost every 2 years. Recent GII.4 variant reported include Lordsdale 1996, Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoon 2007, New Orleans 2009,most recently Sydney 2012. Detailed molecular epidemiologic investigation of NoV is associated for understanding the genetic diversity of NoV strain and emergence of novel NoV variants. However, reports have revealed that not all individuals develop symptoms and a significant proportion remains asymptomatic after NoV infections.

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Biologic, Antigenic, and Full-Length Genomic Characterization of a Bovine-Like Coronavirus Isolated from a Giraffe

Journal of Virology ◽

10.1128/jvi.02361-06 ◽

2007 ◽

Vol 81 (10) ◽

pp. 4981-4990 ◽

Cited By ~ 56

Author(s):

Mustafa Hasoksuz ◽

Konstantin Alekseev ◽

Anastasia Vlasova ◽

Xinsheng Zhang ◽

David Spiro ◽

...

Keyword(s):

Amino Acid ◽

Genetic Relatedness ◽

Phylogenetic Analyses ◽

Point Mutations ◽

The United States ◽

Interspecies Transmission ◽

Adaptive Mutations ◽

Severe Diarrhea ◽

Wild Ruminants

ABSTRACT Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.

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Population Genomics of GII.4 Noroviruses Reveal Complex Diversification and New Antigenic Sites Involved in the Emergence of Pandemic Strains

10.1101/668772 ◽

2019 ◽

Author(s):

Kentaro Tohma ◽

Cara J. Lepore ◽

Yamei Gao ◽

Lauren A. Ford-Siltz ◽

Gabriel I. Parra

Keyword(s):

Amino Acid ◽

Capsid Protein ◽

Large Scale ◽

Population Genomics ◽

Viral Capsid ◽

Viral Gastroenteritis ◽

Viral Capsid Protein ◽

Antigenic Sites ◽

Capsid Protein Vp1

AbstractGII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights on the mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large number (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside of the antigenic sites. Residues from the third pattern formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic make-up of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with two of them (C and G) containing residues (352, 357, 378) linked with the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by a stochastic diversification with minimal changes at the antigenic sites that did not progress towards the next variant. This study provides a methodological framework for antigenic characterization of viruses, and expands our understanding of the dynamics of GII.4 noroviruses that could facilitate the design of cross-reactive vaccines.ImportanceNoroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is an inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design.

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Population Genomics of GII.4 Noroviruses Reveal Complex Diversification and New Antigenic Sites Involved in the Emergence of Pandemic Strains

mBio ◽

10.1128/mbio.02202-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 11

Author(s):

Kentaro Tohma ◽

Cara J. Lepore ◽

Yamei Gao ◽

Lauren A. Ford-Siltz ◽

Gabriel I. Parra

Keyword(s):

Amino Acid ◽

Capsid Protein ◽

Large Scale ◽

Population Genomics ◽

Viral Capsid ◽

Viral Gastroenteritis ◽

Viral Capsid Protein ◽

Antigenic Sites ◽

Capsid Protein Vp1

ABSTRACT GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights into the mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large proportion (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside the antigenic sites. Residues in the third pattern category formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic makeup of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with three of them (A, C, and G) containing residues (352, 357, 368, and 378) linked with the diversifying selective pressure on the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by stochastic diversification with minimal changes that did not progress toward the next variant. This report provides a methodological framework for antigenic characterization of viruses and expands our understanding of the dynamics of GII.4 noroviruses and could facilitate the design of cross-reactive vaccines. IMPORTANCE Noroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design.

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Identification and Characterization of a Carlavirus Causing Veinal Necrosis of Coleus

Plant Disease ◽

10.1094/pdis-91-6-0754 ◽

2007 ◽

Vol 91 (6) ◽

pp. 754-757 ◽

Cited By ~ 9

Author(s):

Dimitre S. Mollov ◽

Maya C. Hayslett ◽

Kari A. Eichstaedt ◽

Noelle G. Beckman ◽

Margery L. Daughtrey ◽

...

Keyword(s):

New York ◽

Amino Acid ◽

Amino Acid Sequence ◽

Capsid Protein ◽

Phylogenetic Analyses ◽

Virus Genome ◽

Coat Protein Region ◽

Veinal Necrosis ◽

Protein Size ◽

Vein Necrosis

A filamentous virus identified in coleus (Coleus × hybrida) in Minnesota and New York was found to cause veinal necrosis in coleus, although this symptom was observed only under certain conditions. The virus was transmitted readily by mechanical inoculation to coleus and Nicotiana spp. and was not transmitted by Myzus persicae. The particles of the coleus virus had a modal length of 640 nm and a single capsid protein with an estimated molecular mass of 34 kDa. The amino acid sequence of the coat protein region of the coleus virus genome had significant similarities only to the corresponding domain of carlaviruses. Based on virion morphology, capsid protein size, genome size and organization, amino acid sequence, and phylogenetic analyses, the coleus virus, which was named provisionally Coleus vein necrosis virus (CVNV), was concluded to be a new definitive member of the genus Carlavirus. A 2-kb fragment of the 3′ terminus of the CVNV genome sequence is accessible under accession number DQ915963 in GenBank.

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phydms: software for phylogenetic analyses informed by deep mutational scanning

PeerJ ◽

10.7717/peerj.3657 ◽

2017 ◽

Vol 5 ◽

pp. e3657 ◽

Cited By ~ 15

Author(s):

Sarah K. Hilton ◽

Michael B. Doud ◽

Jesse D. Bloom

Keyword(s):

Natural Selection ◽

Amino Acid ◽

Gene Evolution ◽

Phylogenetic Analyses ◽

Point Mutations ◽

Quantitative Comparison ◽

Experimental Measurements ◽

Site Specific ◽

Substitution Models ◽

Actual Selection

It has recently become possible to experimentally measure the effects of all amino-acid point mutations to proteins using deep mutational scanning. These experimental measurements can inform site-specific phylogenetic substitution models of gene evolution in nature. Here we describe software that efficiently performs analyses with such substitution models. This software, phydms, can be used to compare the results of deep mutational scanning experiments to the selection on genes in nature. Given a phylogenetic tree topology inferred with another program, phydms enables rigorous comparison of how well different experiments on the same gene capture actual natural selection. It also enables re-scaling of deep mutational scanning data to account for differences in the stringency of selection in the lab and nature. Finally, phydms can identify sites that are evolving differently in nature than expected from experiments in the lab. As data from deep mutational scanning experiments become increasingly widespread, phydms will facilitate quantitative comparison of the experimental results to the actual selection pressures shaping evolution in nature.

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Emergence of Amoxicillin Resistance and Identification of Novel Mutations of the pbp1A Gene in Helicobacter pylori in Vietnam

10.21203/rs.3.rs-1099024/v1 ◽

2021 ◽

Author(s):

Trung Thien Tran ◽

Anh Tuan Nguyen ◽

Duc Trong Quach ◽

Dao Thi-Hong Pham ◽

Nga Minh Cao ◽

...

Keyword(s):

Helicobacter Pylori ◽

Amino Acid ◽

Molecular Mechanisms ◽

Direct Sequencing ◽

Phylogenetic Analyses ◽

Point Mutations ◽

Upper Gastrointestinal Endoscopy ◽

Primary Resistance ◽

H Pylori ◽

Tree Building

Abstract Background Amoxicillin resistant Helicobacter pylori (H. pylori) strains seem to have increased over time in Vietnam. This threatens the effectiveness of H. pylori eradication therapies with this antibiotic. This study aimed to investigate the prevalence of primary resistance of H. pylori to amoxicillin and to assess its association with pbp1A point mutations in Vietnamese patients. Materials and Methods Naive patients who presented with dyspepsia undergoing upper gastrointestinal endoscopy were recruited. Rapid urease tests and PCR assays were used to diagnose H. pylori infection. Amoxicillin susceptibility was examined by E-tests. Molecular detection of the mutant pbp1A gene conferring amoxicillin resistance was carried out by real-time PCR followed by direct sequencing of the PCR products. Phylogenetic analyses were performed using the Tamura-Nei genetic distance model and the neighbour-joining tree building method. Results There were 308 patients (46.1% men and 53.9% women, p = 0.190) with H. pylori infection. The mean age of the patients was 40.5 ± 11.4 years, ranging from 18 to 74 years old. The E-test was used to determine the susceptibility to amoxicillin (minimum inhibitory concentration (MIC) ≤ 0.125 µg/ml) in 101 isolates, among which the rate of primarily resistant strains to amoxicillin was 25.7%. Then, 270 sequences of pbp1A gene fragments were analysed. There were 77 amino acid substitution positions investigated, spanning amino acids 310–596, with the proportion varying from 0.4–100%. Seven amino acid changes were significantly different between amoxicillin-sensitive (AmoxS) and amoxicillin-resistant (AmoxR) samples, including Phe366 to Leu (p < 0.001), Ser414 to Arg (p < 0.001), Glu/Asn464−465 (p = 0.009), Val469 to Met (p = 0.021), Phe473 to Val (p < 0.001), Asp479 to Glu (p = 0.044), and Ser/Ala/Gly595−596 (p = 0.001). Phylogenetic analyses suggested that other molecular mechanisms might contribute to amoxicillin resistance in H. pylori in addition to the alterations in PBP1A. Conclusions We reported the emergence of amoxicillin-resistant Helicobacter pylori strains in Vietnam and new mutations statistically associated with this antimicrobial resistance. Additional studies are necessary to identify the mechanisms contributing to this resistance in Vietnam.

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Epochal Evolution of GGII.4 Norovirus Capsid Proteins from 1995 to 2006

Journal of Virology ◽

10.1128/jvi.00674-07 ◽

2007 ◽

Vol 81 (18) ◽

pp. 9932-9941 ◽

Cited By ~ 274

Author(s):

J. Joukje Siebenga ◽

Harry Vennema ◽

Bernadet Renckens ◽

Erwin de Bruin ◽

Bas van der Veer ◽

...

Keyword(s):

Amino Acid ◽

Phylogenetic Analyses ◽

Three Dimensional ◽

Capsid Proteins ◽

Dimensional Structure ◽

Viral Gastroenteritis ◽

Norwalk Virus ◽

Genetic Changes ◽

Population Immunity ◽

Amino Acid Mutations

ABSTRACT Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains.

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Phylogenetic Analysis: Basic Concepts and Its Use as a Tool for Virology and Molecular Epidemiology

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.81158 ◽

2018 ◽

Vol 44 (1) ◽

pp. 20

Author(s):

Eloiza Teles Caldart ◽

Helena Mata ◽

Cláudio Wageck Canal ◽

Ana Paula Ravazzolo

Keyword(s):

Phylogenetic Analysis ◽

Amino Acid ◽

Molecular Epidemiology ◽

Phylogenetic Analyses ◽

Phylogenetic Reconstruction ◽

Evolutionary Process ◽

Amino Acid Sequences ◽

Evolutionary Models ◽

Reconstruction Methods ◽

Basic Concepts

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology.Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. There are a number of evolutionary models available, varying in complexity according to the number of parameters (transition, transversion, GC content, nucleotide position in the codon, among others). Some papers presented herein provide techniques that can be used to choose evolutionary models. After the model is chosen, the next step is to opt for a phylogenetic reconstruction method that best fits the available data and the selected model. Here we present the most common reconstruction methods currently used, describing their principles, advantages and disadvantages. Distance methods, for example, are simpler and faster, however, they do not provide reliable estimations when the sequences are highly divergent. The accuracy of the analysis with probabilistic models (neighbour joining, maximum likelihood and bayesian inference) strongly depends on the adherence of the actual data to the chosen development model. Finally, we also explore topology confidence tests, especially the most used one, the bootstrap. To assist the reader, this review presents figures to explain specific situations discussed in the text and numerous examples of previously published scientific articles in virology that demonstrate the importance of the techniques discussed herein, as well as their judicious use.Conclusion: The DNA sequence is not only a record of phylogeny and divergence times, but also keeps signs of how the evolutionary process has shaped its history and also the elapsed time in the evolutionary process of the population. Analyses of genomic sequences by molecular phylogeny have demonstrated a broad spectrum of applications. It is important to note that for the different available data and different purposes of phylogenies, reconstruction methods and evolutionary models should be wisely chosen. This review provides theoretical basis for the choice of evolutionary models and phylogenetic reconstruction methods best suited to each situation. In addition, it presents examples of diverse applications of molecular phylogeny in virology.

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Genomic Variations in Drug Resistant Mycobacterium tuberculosis Strains Collected from Patients with Different Localization of Infection

Antibiotics ◽

10.3390/antibiotics10010027 ◽

2020 ◽

Vol 10 (1) ◽

pp. 27

Author(s):

Ekaterina Chernyaeva ◽

Mikhail Rotkevich ◽

Ksenia Krasheninnikova ◽

Alla Lapidus ◽

Dmitrii E. Polev ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Pulmonary Tuberculosis ◽

Central Asia ◽

Phylogenetic Analyses ◽

Treatment Success ◽

Point Mutations ◽

Whole Genome Sequence ◽

Public Health Importance ◽

Resistance Patterns

Mycobacterium tuberculosis is a highly studied pathogen due to public health importance. Despite this, problems like early drug resistance, diagnostics and treatment success prediction are still not fully resolved. Here, we analyze the incidence of point mutations widely used for drug resistance detection in laboratory practice and conduct comparative analysis of whole-genome sequence (WGS) for clinical M. tuberculosis strains collected from patients with pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (XPTB) localization. A total of 72 pulmonary and 73 extrapulmonary microbiologically characterized M. tuberculosis isolates were collected from patients from 2007 to 2014 in Russia. Genomic DNA was used for WGS and obtained data allowed identifying major mutations known to be associated with drug resistance to first-line and second-line antituberculous drugs. In some cases previously described mutations were not identified. Using genome-based phylogenetic analysis we identified M. tuberculosis substrains associated with distinctions in the occurrence in PTB vs. XPTB cases. Phylogenetic analyses did reveal M. tuberculosis genetic substrains associated with TB localization. XPTB was associated with Beijing sublineages Central Asia (Beijing CAO), Central Asia Clade A (Beijing A) and 4.8 groups, while PTB localization was associated with group LAM (4.3). Further, the XPTB strain in some cases showed elevated drug resistance patterns relative to PTB isolates. HIV was significantly associated with the development of XPTB in the Beijing B0/W148 group and among unclustered Beijing isolates.

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