Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles

AbstractThe simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77–93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly

Mosaic Qβ coats as a new presentation model

AbstractThe new protein carrier was developed on the basis of recombinant RNA phage Qβ capsid. C-terminal UGA extension of the short form of Qβ coat, so-called A1 extension, served as a target for presentation of foreign peptides on the outer surface of mosaic Qβ particles. In conditions of enhanced UGA suppression, the proportion of A1-extended to short coats in mosaic particles dropped from 48% to 14%, with an increase of the length of A1 extension. A model insertion, short preS1 epitope 31-DPAFR-35 of hepatitis B surface antigen, demonstrated superficial location on the mosaic Qβ particles and ensured specific antigenicity and immunogenicity

Development of rapid antigen test prototype for detection of SARS-CoV-2 in saliva samples

Funding Information: This study was supported by VPP Program, Republic of Latvia Ministry of Education and Science, project No. VPP-COVID-2020/1-0025. Publisher Copyright: © 2022 Taylor and Francis Ltd. All rights reserved.Background: The development of easy-to-perform diagnostic methods is highly important for detecting current coronavirus disease (COVID-19). This pilot study aimed at developing a lateral flow assay (LFA)- based test prototype to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva samples. Methods: Mice were immunized using the recombinant receptor-binding domain (rRBD) of SARS-CoV-2 virus spike protein. The combinations of the obtained mouse anti-receptor-binding domain (RBD) polyclonal antibodies (PAbs) and several commercial antibodies directed against the SARS-CoV-2 spike protein were used for enzyme-linked immunosorbent assay (ELISA) to select antibody pairs for LFA. The antibody pairs were tested in a LFA format using saliva samples from individuals with early SARS-CoV-2 infection (n = 9). The diagnostic performance of the developed LFA was evaluated using saliva samples from hospitalized COVID-19 patients (n = 111); the median time from the onset of symptoms to sample collection was 10 days (0-24 days, interquartile range (IQR): 7-13). The reverse transcription-polymerase chain reaction (rRT-PCR) was used as a reference method. Results: Based on ELISA and preliminary LFA results, a combination of mouse anti-RBD PAbs (capture antibody) and rabbit anti-spike PAbs (detection antibody) was chosen for clinical analysis of sample. When compared with rRT-PCR results, LFA exhibited 26.5% sensitivity, 58.1% specificity, 50.0% positive prediction value (PPV), 33.3% negative prediction value (NPV), and 38.7% diagnostic accuracy. However, there was a reasonable improvement in assay specificity (85.7%) and PPV (91.7%) when samples were stratified based on the sampling time. Conclusion: The developed LFA assay demonstrated a potential of SARS-CoV-2 detection in saliva samples. Further technical assay improvements should be made to enhance diagnostic performance followed by a validation study in a larger cohort of both asymptomatic and symptomatic patients in the early stage of infection.Peer reviewe

Structural study of the catalytic domain of PKCfusinginfrared spectroscopy and two-dimensional infraredcorrelation spectroscopy

The secondary structure of the catalytic domain from protein kinase Cfwas studied using IR spectroscopy. In the presence of the substrateMgATP, there was a significant change in the secondary structure. Afterheating to 80°C, a 14% decrease in thea-helix component was observed,accompanied by a 6% decrease in theb-pleated sheet; no change wasobserved in the large loops or in 310-helix plus associated loops. The maxi-mum increase with heating was observed in the aggregatedb-sheet compo-nent, with an increase of 14%. In the presence of MgATP, and comparedwith the sample heated in its absence, there was a substantial decrease inthe 310-helix plus associated loops and an increase ina-helix. Synchronous2D-IR correlation showed that the main changes occurred at 1617 cm)1,which was assigned to changes in the intermolecular aggregatedb-sheet ofthe denaturated protein. This increase was mainly correlated with thechange ina-helix. In the presence of MgATP, the main correlation wasbetween aggregatedb-sheet and the large loops component. The asynchro-nous 2D-correlation spectrum indicated that a number of components aretransformed in intermolecularly aggregatedb-sheet, especially thea-helixandb-sheet components. It is interesting that changes in 310-helix plusassociated loops and ina-helix preceded changes in large loops, which sug-gests that the open loops structure exists as an intermediate state duringdenaturation. In summary, IR spectroscopy revealed an important effect ofMgATP on the secondary structure and on the thermal unfolding processwhen this was induced, whereas 2D-IR correlation spectroscopy allowed usto show the establishment of the denaturation pathway of this proteinMedicin

Mozaikalu virusveidigu dalinu konstruesana un struktura

Due to unique spatial structure and self-assembly properties, hepatitis B virus core antigen (HBcAg) and its C-terminally truncated form (HBc#DELTA#) have been widely used as carriers to incorporate and expose foreign protein sequences on their surface. A new strategy was developed for insertions into the C-terminus of HBc#DELTA#, which is based on introduction of stop-codon between the HBc#DELTA# and sequences encoding foreign protein. Simultaneous synthesis of wild-type (wt) HBc#DELTA# as a helper and wt/fusion monomers was detected from single expression cassette in conditions of stop-codon suppression. Incorporation of 93, 114, and 213 aa of hantavirus nucleoprotein into mosaic HBc particles was demonstrated, whereas "pure" HBc#DELTA#hanta fusion proteins expressed alone were unable to form particles or formed them at low extent. Purified mosaic particles elicited both anti-HBc and anti-hanta response in mice. Alternatively, two-replicon based system was developed to ensure co-expression of two HBc-derived proteins from separate plasmids in the same E. coli cell. Applicability of this system was evaluated with full-length HBV preS sequence inserted at the C-terminus of the HBc#DELTA#. After co-expression of the HBc#DELTA#preS fusion with the wt HBc#DELTA# as a helper, synthesis of both proteins was observed. Presence of the wt HBc#DELTA# significantly improved solubility of the HBc#DELTA#preS fusion, which was mainly insoluble, when expressed alone. Purified particles contained both wt and HBc#DELTA#preS fusion monomers and demonstrated high anti-preS antigenicity in ELISA tests. The same approach was used to investigate possible interaction of wt and in-frame HBc deletion mutants occurring in HBV-infected renal transplantation patients. Synthesis and solubility of assembly non-competent 17 aa deletion mutant was markedly enhanced in the presence of the wt HBc. Immunoprecipitation of particles with monoclonal antibody recognizing sequence, which is deleted in mutant, allowed to detect incorporation of the mutant protein into mosaic HBc particles. A novel method to investigate dimeric content and composition within mosaic particles was developed. This method is based on fixation of disulfide bridges between monomers, with following disruption of particles in non-reducing conditions. It allowed to demonstrate both homo- and heterodimers within mosaic particles. Relative proportions of homo- and heterodimers varied in mosaic particles. The results of this work are reflected in six papers (one of them in manuscript) and reported at three international workshops as well as at the numerous scientific conferences of the University of LatviaSummary in LatvianAvailable from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

Genome Characterization of Nocturne116, Novel Lactococcus lactis-Infecting Phage Isolated from Moth

While looking for novel insect-associated phages, a unique siphophage, Nocturne116, was isolated from a deceased local moth specimen along with its host, which was identified by 16S rRNA gene sequencing as a strain of Lactococcus lactis. Next-generation sequencing and the subsequent genome annotation elaborated on herein revealed that the genome of Nocturne116 is a 25,554 bp long dsDNA molecule with 10 bp long 3′ cos overhangs and a GC content of 37.99%, comprising 52 predicted open reading frames. The complete nucleotide sequence of phage Nocturne116 genome is dissimilar to any of the already sequenced phages, save for a distant link with Lactococcus phage Q54. Functions for only 15/52 of Nocturne116 gene products could be reliably predicted using contemporary comparative genomics approaches, while 22 of its gene products do not yet have any homologous entries in the public biological sequence repositories. Despite the public availability of nearly 350 elucidated Lactococcus phage complete genomes as of now, Nocturne116 firmly stands out as a sole representative of novel phage genus

Morganella Phage Mecenats66 Utilizes an Evolutionarily Distinct Subtype of Headful Genome Packaging with a Preferred Packaging Initiation Site

Both recognized species from the genus Morganella (M. morganii and M. psychrotolerans) are Gram-negative facultative anaerobic rod-shaped bacteria that have been documented as sometimes being implicated in human disease. Complete genomes of seven Morganella-infecting phages are publicly available today. Here, we report on the genomic characterization of an insect associated Morganella sp. phage, which we named Mecenats66, isolated from dead worker honeybees. Phage Mecenats66 was propagated, purified, and subjected to whole-genome sequencing with subsequent complete genome annotation. After the genome de novo assembly, it was noted that Mecenats66 might employ a headful packaging with a preferred packaging initiation site, although its terminase amino acid sequence did not fall within any of the currently recognized headful packaging subtype employing phage (that had their packaging strategy experimentally verified) with clusters on a terminase sequence phylogenetic tree. The in silico predicted packaging strategy was verified experimentally, validating the packaging initiation site and suggesting that Mecenats66 represents an evolutionarily distinct headful genome packaging with a preferred packaging initiation site strategy subtype. These findings can possibly be attributed to several of the phages already found within the public biological sequence repositories and could aid newly isolated phage packaging strategy predictions in the future

Three Phages One Host: Isolation and Characterization of <i>Pantoea agglomerans</i> Phages from a Grasshopper Specimen

The bacterial genus Pantoea comprises species found in a variety of different environmental sources. Pantoea spp. are often recovered from plant material and are capable of both benefitting the plants and acting like phytopathogens. Some species of Pantoea (including P. agglomerans) are considered opportunistic human pathogens capable of causing various infections in immunocompromised subjects. In this study, a strain of P. agglomerans (identified by 16S rRNA gene sequencing) was isolated from a dead specimen of an unidentified Latvian grasshopper species. The retrieved strain of P. agglomerans was then used as a host for the potential retrieval of phages from the same source material. After rounds of plaque purification and propagation, three high-titer lysates corresponding to putatively distinct phages were acquired. Transmission electron microscopy revealed that one of the phages was a myophage with an unusual morphology, while the two others were typical podophages. Whole-genome sequencing (WGS) was performed for each of these isolated phages. Genome de novo assembly and subsequent functional annotation confirmed that three different strictly lytic phages were isolated. Elaborate genomic characterization of the acquired phages was performed to elucidate their place within the so-far-uncovered phage diversity

Three Phages One Host: Isolation and Characterization of Pantoea agglomerans Phages from a Grasshopper Specimen

The bacterial genus Pantoea comprises species found in a variety of different environmental sources. Pantoea spp. are often recovered from plant material and are capable of both benefitting the plants and acting like phytopathogens. Some species of Pantoea (including P. agglomerans) are considered opportunistic human pathogens capable of causing various infections in immunocompromised subjects. In this study, a strain of P. agglomerans (identified by 16S rRNA gene sequencing) was isolated from a dead specimen of an unidentified Latvian grasshopper species. The retrieved strain of P. agglomerans was then used as a host for the potential retrieval of phages from the same source material. After rounds of plaque purification and propagation, three high-titer lysates corresponding to putatively distinct phages were acquired. Transmission electron microscopy revealed that one of the phages was a myophage with an unusual morphology, while the two others were typical podophages. Whole-genome sequencing (WGS) was performed for each of these isolated phages. Genome de novo assembly and subsequent functional annotation confirmed that three different strictly lytic phages were isolated. Elaborate genomic characterization of the acquired phages was performed to elucidate their place within the so-far-uncovered phage diversity