Caspian Sandy Natural Focus: Phylogenetic History and Origin of <i>Yersinia pestis</i> Strains

The purpose of the work was to analyze the phylogenetic relations and origin of Yersinia pestis strains isolated in different periods of epizootic activity of the Caspian sandy natural focus (CSNF) of plague in the XX–XXI centuries.Materials and methods. We used 40 Y. pestis strains from CSNF and adjacent plague foci, isolated in 1922–2015. Carried out was whole genome sequencing of 19 Y. pestis strains from CSNF. Phylogenetic analysis was performed using whole genome SNP analysis based on 1914 identified SNPs. The search for marker SNPs was conducted using the Snippy 4.6 software. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model.Results and discussion. The whole genome SNP analysis has revealed that Y. pestis strains of the medieval biovar from CSNF belong to 2.MED1 phylogenetic lineage and fall into two major branches. One of them circulated in the focus in the first half of the XX century, and the other – in the second half of the XX – early XXI centuries. It is shown that strains of the first branch were the cause of outbreaks and individual cases of plague in the CSNF in the first half of the XX century. They are closely related to strains from the Caspian North-Western steppe and Volga-Ural sandy natural plague foci, which caused numerous outbreaks with high mortality rate in the same period. Y. pestis strains from the CSNF of the second half of the XX and early XXI centuries belong to the second phylogenetic branch of the 2.MED1 line, at the node of which the strains from the Northern Aral Sea region of 1945 lay. The latter were the predecessors of all strains isolated in the CSNF after a long inter-epizootic period that occurred in the middle of the XX century. There can also be traced a genetic relation between the strains from CSNF and the Dagestan plain-foothill focus

Variability of <i>pgm</i>‑Region Genes in <i>Yersinia pestis</i> Strains from the Caspian Sandy and Adjacent Plague Foci

The aim of the study was to compare the nucleotide sequences of pgm‑region genes in Yersinia pestis strains isolated on the territory of the Caspian sandy and adjacent plague foci in 1925–2015. Materials and methods. 65 Y. pestis strains from the Caspian sandy and adjacent plague foci were used in the work. DNA isolation was performed using the PureLink Genomic DNA Mini Kit. Whole genome sequencing was conducted in Ion S5 XL System (Thermo Fischer Scientific). Data processing was carried out using Ion Torrent Suite software package 3.4.2 and NewblerGS Assembler 2.6. To compare the obtained sequences with the NCBI GenBank database, the Blast algorithm was used. The phylogenetic analysis was performed according to the data of whole genome SNP analysis based on 1183 identified SNPs. The search for marker SNPs was performed using the Snippy 4.6 program. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model. Results and discussion. The nucleotide sequences of pgm‑region genes of 65 Y. pestis strains from the Caspian sandy and adjacent plague foci have been assessed. Single nucleotide substitutions have been identified in Y. pestis strains from the Caspian sandy and Kobystan plain-foothill foci in the hmsR, astB, ybtS, ypo1944, ypo1943, ypo1936 genes, as well as a deletion of 5 bp in the ypo1945 gene, which is characteristic of strains of one of the phylogenetic lines of Y. pestis from the foci of Caucasus and Transcaucasia, isolated in 1968–2001. The data obtained can be used to differentiate Y. pestis strains from the Caspian sandy focus, as well as to establish the directions of microevolution of the plague pathogen in this region and adjacent foci

Long-Term Persistence of <i>Yersinia pestis</i> in Association with Acanthamoeba castellanii in Experiment

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods

Improvement of Approaches to the Verification of the Vaccine Strain <i>Francisella tularensis</i> 15 NIIEG during Long-Term Storage

The aim of the study was to improve the methods for verifying the vaccine strain Francisella tularensis 15 NIIEG during long-term storage under current conditions.Materials and methods. The paper summarizes the results of studying the phenotypic and genetic properties of lyophilized cultures of the vaccine strain F. tularensis 15 NIIEG (1953, 1966, 1969, 1987, 1990, 2003, 2012 and 2013) stored at SCEMAP for a period of one to 60 years.Results and discussion. Previous studies have revealed that freeze-dried cultures of F. tularensis 15 NIIEG generally had the characteristics of the vaccine strain, with the exception of deviations from the regulatory requirements for residual virulence and specific safety. The stability of preservation of deletions in the pilA and pilE genes (the region of differentiation RD19) and the genes encoding lpp lipoprotein (RD18) in the vaccine strain, which was stored for various periods of time in a lyophilized state, has been confirmed. The vaccine-strain-specific mutation C178404T (by the genome of F. tularensis LVS strain, GenBank NCBI no. CP009694) has been identified, and an approach to determine it has been developed. The data obtained are promising as regards using the above deletions in the RD18/RD19 regions in combination with the C178404T mutation to assess the authenticity of the vaccine strain using molecular genetic methods. Thus, the conducted retrospective analysis of the data on the cultures of tularemia microbe vaccine strain from the 1940s to 2013 and the gathered experimental data, made it possible to supplement the uniform requirements for the manufacture, study, maintenance, storage and movement of F. tularensis 15 NIIEG vaccine strain with new evidence. Based on the results obtained, the authors have drawn a draft methodological recommendations of the federal level “Vaccinal strain Francisella tularensis 15 NIIEG: order of handling”

Tracing the Spatial Circulation of Yersinia pestis of Medieval Biovar in the Eastern Caspian Sea Region in the 20th Century Based on Genome-Wide SNP Analysis

A comprehensive analysis of the accumulated epidemiological and epizootiological data in combination with results from phylogenetic analysis of Yersinia pestis strains creates the basis for establishing patterns of spatialtemporal distribution of the plague pathogen and opens up the prospect of long-term forecasting of natural plague foci activation. Previously, we traced the distribution pathways of Y. pestis, medieval biovar, in the plague foci of the Northern and Northwestern Caspian Sea regions in the 20th and early 21st centuries.The purpose of this work was to identify the regularities of circulation of Y. pestis, medieval biovar, in four natural plague foci located in the Eastern Caspian Sea region.Materials and methods. A complex study of the phenotypic and genetic properties of 16 Y. pestis strains isolated in the Ustyurt, Mangyshlak, Karakum and Kopetdag autonomous desert plague foci in 1926–1985 was carried out. They were compared with strains from other natural plague foci in Eastern Europe and Central Asia obtained in 1917–2003. Whole-genome sequencing of 12 of those strains was performed. Phylogenetic analysis included the genomes of other 19 Y. pestis strains that we had sequenced earlier. Based on the 1717 polymorphic nucleotides (SNPs) identified in the core genome, a dendrogram of the relations of the studied strains was constructed.Results and discussion. All 16 Y. pestis strains from the Ustyurt, Mangyshlak, Kopetdag, and Karakum desert foci belong to the 2.MED1 branch of the medieval biovar. All investigated strains from the first three foci and most of the strains from the Karakum focus are in the Caspian 2.MED1 branch, and three strains from the Karakum desert focus are included in the Central Asian one. We have revealed several waves of dissemination of the strains under the 2.MED1 phylogenetic branch of Y. pestis of the medieval biovar in the Eastern Caspian Sea region in the 20th century

Spatial Structure of <i>Yersinia pestis</i> Population Belonging to Altai Biovar, Subspecies central asiatica Acording to Genome-Wide Sequencing Data

The aim of the work was to conduct phylogenetic analysis of Y. pestis strains, biovar altaica ssp. central asiatica, isolated in Gorno-Altai high-mountain and Sailyugem natural plague foci on the territory of Russia and Mongolia in 1965–2020, by full-genome sequencing data.Materials and methods. To determine the population structure of the biovar altaica ssp. central asiatica, 34 whole genome sequences were used (including 20 Y. pestis strains of the biovar altaica, 18 of which were sequenced by us). To isolate DNA from the Y. pestis strains, a PureLink Genomic DNA Mini Kit (Invitrogen, USA) was applied. Sequencing of the nucleotide sequences of Y. pestis strains was carried out in Ion PGM system Lifetechnologies. The analysis and processing of the obtained data were performed with the help of Newblergs Assembler 2.6 and IonTorrent Suite software package, 3.4.2. The search for SNPs was performed using the Wombac 2.0 program. The Maximum Likelihood dendrogram was built in the PhyML 3.1. The dendrogram was visualized using the FigTree 1.4.3 software.Results and discussion. Based on the data of whole genome analysis, taking into account the 1871 revealed polymorphic nucleotides, the spatial structure of the biovar altaica ssp. central asiatica has been determined. It includes several phylogeographic branches: the Kurai-Tarkhatinskaya (cluster 0.PE4a-1) and the Ulandryk-Mongolian (0.PE4a-2), which is in agreement with the geographical regions of the isolation of strains forming these branches in the Altai Mountains. The Kurai-Tarkhatinskaya branch is further divided into the Kurai (sub-cluster 0.PE4a-1-1, formed by the strains of 2009–2018) and Tarkhatinskaya (subcluster 0.PE4a-1-2, formed by the strains of 2012–2020) sub-branches, while the Ulandryk-Mongolian branch of evolution is split into sub-branches represented by strains from the Ulandryk meso focus (sub-cluster 0.PE4a-2-2, strains 1965–2010) and the Sailyugem focus of Mongolia (sub-cluster 0.PE4a-2-1, strains 1964–1990)

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Peripheral lymph node tuberculosis: its paradoxical course in a HIV-infected patient

Периферическая лимфатическая система инфицируется микобактерией туберкулеза (МБТ) во время лимфогенной диссеминации, но не у всех пациентов развивается клинически документированное поражение лимфоузлов