Molecular Genetics, Microbiology and Virology最新文献

Abstract

Bacterial ghosts (BGs) are gram-negative bacteria cell membranes without cytoplasmic content obtained by lysis as a result of cell-wall perforation mediated by the φX174 bacteriophage E protein. Introducing into a lytic plasmid lysis gene from other bacteriophages, which target alternative molecular targets, in addition to the E protein gene of bacteriophage φX174, can lead to an increase in the efficiency of lysis and, in some cases, to an increase in the immunogenicity of preparations. Escherichia coli strains carrying plasmids with various combinations of genes encoding protein E of phage φ174 with cassettes of lytic genes of the “choline–endolysin” systems of phages λ or L-413C, providing different degrees of destruction of the cell wall, were obtained for the subsequent selection of the most promising lytic structures. The formation of E. coli ghosts and the release of cell contents was confirmed by transmission electron microscopy. When the cultivation temperature was increased from 28 to 42°C, the lysis of E. coli cultures DH5α/pEYR'-E, DH5α/pEYR'-Y-K, DH5α/pEYR'-E-Y-K, and DH5α/pEYR'-E-Sam7-R-Rz was observed. Lysis was practically not detected during the growth of the DH5α/pEYR'-S-R-Rz culture, as in the control strain DH5α/pEYR'. The results of the analysis of ultrathin sections of BGs preparations by transmission electron microscopy (TEM) were comparable with the data on optical density and the results of inoculation of induced cultures of engineered strains, and also made it possible to assess the fine structure of bacterial cells carrying various combinations of lytic phage genes. BGs of gram-negative bacteria are promising for the creation of highly effective inactivated candidate vaccines that could replace currently available bacterial heat-inactivated and formol vaccines.

Hepatitis B is a major public health problem worldwide. The indigenous population of remote regions with harsh living conditions, such as the Far North of Russia, with possibly unique genetic mutations in the local strains of this pathogen, remains poorly studied. The aim of the study was to determine the prevalence of serological HBV markers in blood–plasma samples of the population of the Far North of Russia and to determine HBV genotypes and HBsAg subtypes in blood–plasma samples containing HBsAg. We studied 702 blood–plasma samples taken from the indigenous population (Tundra Nenets) of the village of Gyda in the Tazovsky district of the Yamalo-Nenets autonomous okrug (YaNAO) (north of the Gydan Peninsula). Detection of serological HBV markers—HBsAg, anti-HBs, and anti-Hbcore—was carried out using JSC Vector-Best reagent kits. HBV genotypes and HBsAg subtypes in the samples were determined according to our own original procedure with monoclonal antibodies and by classical molecular-biological methods. The prevalence of HBsAg in the entire sample was 0.4% (3/702). For the subgroup of samples (persons older than 15 years during the first two expeditions, 300 samples), an extended analysis of the markers was carried out. The following frequencies were determined for them: HBsAg, 0.7% (2/300); anti-HBs, 63.7% (191/300); and anti-HBcore, 11.3% (34/300). HBV statuses were established for this subgroup: the presence of HBV infection (0.7%), past hepatitis B infection (9.3%), and immunity as result of vaccination (54.3%). Genotype D was established for two samples containing HBsAg (subtypes ayw2 and ayw3); the results of the two used methods coincided. The prevalence of HBsAg in the Tazovsky and adjacent Purovsky districts (the main population being Nenets, previously published data) differed (0.4% vs. 1.7%, p < 0.05), presumably due to vaccination (samples of the Purovsky district were collected almost 20 years earlier), as well as the distribution of HBV genotypes. The Tazovsky district of YaNAO is now a low endemic region in terms of HBV infection. The HBV isolates belong to the most common HBV genotype D in the Russian Federation (HBsAg subtypes ayw2, ayw3).

Abstract

Approximately 90% of the world’s population is infected with the Epstein–Barr virus (EBV). Damage to health and high economical costs require the development of vaccines against EBV. The variability of individual genes may affect the success of the vaccination campaign and will necessitate dynamic update of antigenic composition of vaccines. The aim of this study was to assess the variability of EBV gp350 and EBNA2 genes isolated from saliva of dental clinic personnel in Moscow oblast. Biosamples were obtained from 105 employees at four dental clinics. For EBV DNA-positive samples, the EBNA2 and gp350 genes were amplified using nested PCR. The nucleotide sequence of gp350 gene was determined during sequencing. A maximum likelihood phylogenetic tree was constructed from the N-terminal fragment of gp350 protein, using B95-8 strain as a reference genome. Phylogenetic analysis also included 222 EBV samples from the NCBI database. The EBNA2 genotype and the whole gp350 gene sequence were determined for 31 DNA samples. Based on the phylogenetic analysis of gp350 protein, the Russian virus population was uniformly distributed along the tree. Meanwhile, 30 samples fell into the Aa clade (A, genotype A for EBNA2 gene; a, similarity of the gp350 sequence with B95-8 [NC_007605.1]) and one sample belonged to the Bb clade (B, genotype B for EBNA2; b , similarity of gp350 with Jijoye [LN827800.1]). For 30 Russian samples of Aa genotype, 22 individual profiles and 16 unique mutations were found. Identical gp350 profiles were found amongst staff working in close professional contact. The identified features indicated the need for further phylogenetic studies of samples collected in Russia to develop and introduce vaccines and monitor their efficiency.