The core proteins (HBc) of the hepatitis B virus (HBV) genotypes A, B, C, D, E, F, and G were cloned and expressed in Escherichia coli (E. coli), and HBc-formed virus-like particles (VLPs) were purified with ammonium sulfate precipitation, gel filtration, and ion exchange chromatography (IEX). The best VLP yield was found for the HBc of the HBV genotypes D and G. For the HBc of the HBV genotypes D, F, and G, the possibility of dissociation and reassociation maintaining the native HBc structure was demonstrated. Single-stranded (ss) and double-stranded (ds) ribonucleic acid (RNA) was successfully packed into HBc VLPs for the HBV genotypes D and G.

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.

The available HBV vaccines based on the HBV surface protein are manufactured in yeasts and demonstrate excellent prophylactic but no therapeutic activity and are thus ineffective against chronic HBV infection. Five different HBV core proteins (HBc)—full length and C-terminally truncated—were used for the insertion of the short, preS1,aa 20–47 and long, preS1phil, aa 12–60 + 89–119 fragments. Modified virus-like particles (VLPs) were compared for their biotechnological and immunological properties. The expression level of HBc-preS1 proteins was high for all investigated proteins, allowing us to obtain 10–20 mg of purified VLPs from a gram of biomass with the combination of gel filtration and ion-exchange chromatography to reach approximately 90% purity of target proteins. The immunogenicity of chimeric VLPs was tested in BALB/c mice, showing a high anti-preS1 response and substantial T-cell proliferation after stimulation with HBc protein. Targeted incorporation of oligonucleotide ODN 1668 in modified HBc-preS1 VLPs was demonstrated.

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.

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage—JELG-KS1—infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture.

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.