Amazing Discoveries of Benthic Fauna from the Abyssal Zone of Lake Baikal.

Lake Baikal is a natural laboratory for the study of species diversity and evolution, as a unique freshwater ecosystem meeting the all of the main criteria of the World Heritage Convention. However, despite many years of research, the true biodiversity of the lake is clearly insufficiently studied, especially that of deep-water benthic sessile organisms. For the first time, plastic waste was raised from depths of 110 to 190 m of Lake Baikal. The aim of this study was to examine the biological community inhabiting the plastic substrate using morphological and molecular genetic analysis. Fragments of plastic packaging materials were densely populated: bryozoans, leeches and their cocoons, capsules of gastropod eggs, and turbellaria cocoons were found. All the data obtained as a result of an analysis of the nucleotide sequences of the standard bar-coding fragment of the mitochondrial genome turned out to be unique. Our results demonstrate the prospects for conducting comprehensive studies of artificial substrates to determine the true biodiversity of benthos in the abyssal zone of Lake Baikal.

New insights into the organisation of the oxidative phosphorylation system in the example of pea shoot mitochondria.

The physical and functional organisation of the OXPHOS system in mitochondria in vivo remains elusive. At present, different models of OXPHOS arrangement, representing either highly ordered respiratory strings or, vice versa, a set of randomly dispersed supercomplexes and respiratory complexes, have been suggested. In the present study, we examined a supramolecular arrangement of the OXPHOS system in pea shoot mitochondria using digitonin solubilisation of its constituents, which were further analysed by classical BN-related techniques and a multidimensional gel electrophoresis system when required. As a result, in addition to supercomplexes I1III2, I1III2IVn and III2IV1-2, dimer V2, and individual complexes I-V previously detected in plant mitochondria, new OXPHOS structures were also revealed. Of them, (1) a megacomplex (IIxIIIyIVz)n including complex II, (2) respirasomes I2III4IVn with two copies of complex I and dimeric complex III2, (3) a minor new supercomplex IV1Va2 comigrating with I1III2, and (4) a second minor form of ATP synthase, Va, were found. The activity of singular complexes I, IV, and V was higher than the activity of the associated forms. The detection of new supercomplex IV1Va2, along with assemblies I1III2 and I1-2III2-4IVn, prompted us to suggest the occurrence of in vivo oxphosomes comprising complexes I, III2, IV, and V. The putative oxphosome's stoichiometry, historical background, assumed functional significance, and subcompartmental location are discussed herein.

The Phylogenetic Position of Spironucleus sp. (Diplomonadida: Hexamitidae) from the Intestine of Chinese Sleeper Perccottus glenii Dybowski, 1877 (Actinopterygii: Odontobutidae).

INTRODUCTION: The Chinese (Amur) sleeper (Perccottus glenii Dybowski, 1877) (Actinopterygii: Odontobutidae) is a freshwater fish species with high invasive potential. Diplomonads have been detected in the intestines of Chinese sleepers using light microscopy. AIM: The aim of this study was to identify the diplomonads in Chinese sleepers using molecular-genetic methods. MATERIALS AND METHODS: The fish used in this analysis were caught in the following bodies of water in Russia between 2014 and 2016: Lake Dolgoe, the floodplain of the Ingoda River (Amur River basin), the Tsna River (the Oka River basin), and the littoral of the Kotlin Island (Gulf of Finland). Partial sequences of small subunit rRNA genes were obtained for the intestinal diplomonads of Chinese sleeper. RESULTS: The analysis of all sequenced samples revealed the presence of Spironucleus salmonis Moore, 1922; other Spironucleus species were not found in the sampled fish. With 82% probability, the sampled sequences of diplomonads from Chinese sleeper formed a separate cluster in the clade of S. salmonis on the phylogenetic tree. CONCLUSION: This is the first record of S. salmonis in fish in the family Odontobutidae.

Characteristics of far eastern strains of tick-borne encephalitis virus.

A comparative study of biological, molecular and genetic characteristics of a collection of ten strains of tick-borne encephalitis virus (TBEV) isolated in Primorsky Krai before 1960 and stored in a lyophilized state for a prolonged period (over 65 years) is presented. The collection includes the Sofjin strain isolated from the brain of a fatal case in Primorsky Krai in 1937 and transferred to the Scientific Research Institute of Epidemiology and Microbiology (Vladivostok) in 1953. All lyophilized viral strains demonstrated great preservation and high infectious activity in the model of 2-day-old non-inbred mice. Whole-genome sequencing showed that all strains belong to the Far East TBEV subtype, comprising three clusters of Sofjin-, Oshima- and Senzhang-like strains. We show that SofjinPYB, Sofjin (Vector) and Sofjin-HO strains form a separate branch of the phylogenetic tree and are closely related to Khabarovsk-Obor-4, but not to the original Sofjin strain. The Sofjin-1953, Sofijin-Chumakov, SofjinKSY and SofjinCDC strains are genetically close to each other and can be used as reference strains for comparative analysis of the tick-borne encephalitis virus population.

Louping ill virus (LIV) in the Far East.

This study focused on finding, culturing, and identifying the biological and genetic characteristics of three louping ill virus (LIV) strains in the south of the Russian Far East. The Primorye-155-77 and Primorye-20-79 virus strains were isolated from Ixodes persulcatus ticks, and the Primorye-185-91 strain was isolated from the blood of a person after a tick bite. According to the hemagglutination and neutralization tests, Primorye-155-77, Primorye-20-79 and Primorye-185-91 had weak reactivity with antibodies in an antiserum against tick-borne encephalitis virus. In Primorye-155-77 and Primorye-20-79, the sequences of the 5' ends of the 2456-nucleotide-long viral RNA including the 5' untranslated region (UTR) and genes of the capsid protein, prM protein and envelope E protein were determined. The complete genome sequence of Primorye-185-91 was determined. The E protein gene of the Negishi strain differed from those of three analyzed strains, as there were mutations resulting in the replacement of three amino acids: Ala163Thr, Asp193Asn and Ala313Thr. The homology of Primorye-185-91 to LIV 369/T2 was 97.57 %, and to the Penrith strain, it was 98.36 %. Phylogenetic analysis showed that Primorye-155-77, Primorye-20-79 and Primorye-185-91 are related to LI/A and LI/K strains isolated in England and Scotland and to the Negishi strain; these strains have a common progenitor. Negishi-like strains were represented by one subtype of louping ill virus, i.e. the British subtype (LIV-Brit). The possibility is discussed of a single introduction of the virus to the Far Eastern region (Japan and Primorsky Krai) from a single natural locus more than 50 years ago.

The nature of replication of tick-borne encephalitis virus strains isolated from residents of the Russian Far East with inapparent and clinical forms of infection.

We describe the biological properties and molecular characteristics of complete genomes of 33 tick-borne encephalitis virus (TBEV) strains that induced different forms of infection, from inapparent to severe focal ones resulting in fatal outcome. Hemagglutinating activity of Oshima-like strains was higher at pH 5.8, while activity of Sofjin- and Senhzang-like strains were higher at pH 6.2 and 6.8, respectively. We determined susceptibility of porcine kidney (PK) cell cultures to these TBEV strains by cytopathic effect (CPE), plaque formation, and size of plaques. The clinical TBEV strains had higher virus titers both in tissue culture infectious dose 50(TCID50) and in plaque-forming unit (PFU) titers and larger plaques than the inapparent strains. A comparison of virus multiplication kinetics by PFU in culture fluid with kinetics of ELISA antigen and hemagglutinin accumulation suggested a different mechanism of interaction between these virus strains and PK cells at the initial stage of cell infection.

The relationship between the structure of the tick-borne encephalitis virus strains and their pathogenic properties.

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.

Comprehensive assessment of the genetics and virulence of tick-borne encephalitis virus strains isolated from patients with inapparent and clinical forms of the infection in the Russian Far East.

We analyzed the genetics and virulence of 35 strains of TBEV isolated from patients with different forms of the infection living in the southern Far East region of Russia. The results of moleculargenetics studies of the TBEV strains showed that most of the strains that cause inapparent infections form a single cluster (I) with the Oshima 5-10 strain from Japan on the phylogenetic tree. A comparison of the amino acid sequences of the viral polyproteins of the studied strains identified 17 amino acid residues distributed unevenly across the polyprotein that distinctly differed between the clusters of inapparent and virulent strains. We detected additional substitutions in the NS1 and NS5 proteins. These substitutions might influence the pathogenic potential of the strains. Using a model of inbred mice of different ages, we examined the virulence of these strains and showed the different pathogenic potentials of strains belonging to different clusters.

NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus.

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20 ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.

Application of Spiroplasma melliferum proteogenomic profiling for the discovery of virulence factors and pathogenicity mechanisms in host-associated spiroplasmas.

To date, no genome of any of the species from the genus Spiroplasma has been completely sequenced. Long repetitive sequences similar to mobile units present a major obstacle for current genome sequencing technologies. Here, we report the assembly of the Spiroplasma melliferum KC3 genome into 4 contigs, followed by proteogenomic annotation and metabolic reconstruction based on the discovery of 521 expressed proteins and comprehensive metabolomic profiling. A systems approach allowed us to elucidate putative pathogenicity mechanisms and to discover major virulence factors, such as Chitinase utilization enzymes and toxins never before reported for insect pathogenic spiroplasmas.

Core proteome of the minimal cell: comparative proteomics of three mollicute species.

Mollicutes (mycoplasmas) have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity. Thus, they may serve as a model of a 'minimal cell': a cell with the lowest possible number of genes yet capable of autonomous self-replication. We present the results of a comparative analysis of proteomes of three mycoplasma species: A. laidlawii, M. gallisepticum, and M. mobile. The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells. They include replication, transcription, translation, and minimal metabolism. The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery. We also obtained a genome core of the respective organisms and compared it with the proteome core. It was found that the genome core encodes 73 more proteins than the proteome core. Apart of proteins which may not be identified due to technical limitations, there are 24 proteins that seem to not be expressed under the optimal conditions.

The acylation state of surface lipoproteins of mollicute Acholeplasma laidlawii.

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.