Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers.

BackgroundEleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications.ResultsRecombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25–35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not.ConclusionsThe yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed.

Many high-income countries have met the SARS-CoV-2 pandemic with overwhelming sequencing resources and have identified numerous distinct lineages, including some with notably altered biology. Over a year into the pandemic following unprecedented reductions in worldwide human mobility, distinct introduced lineages of SARS-CoV-2 without sequenced antecedents are increasingly discovered in high-income countries as a result of ongoing SARS-CoV-2 genomic surveillance initiatives. We here describe one such SARS-CoV-2 lineage, carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69del, Y144del, and LLA241/243del. This lineage - designated B.1.620 - is known to circulate in Lithuania and has now been found in several European states, but also in increasing numbers in central Africa owing to important recent increases in genome sequencing efforts on the continent. We provide evidence of likely ongoing local transmission of B.1.620 in Lithuania, France, Germany, Spain, Belgium and the Central African Republic. We describe the suite of mutations this lineage carries, its potential to be resistant to neutralising antibodies, travel histories for a subset of the European cases, and evidence of local B.1.620 transmission in Europe. We make a case for the likely Central African origin of this lineage by providing travel records as well as the outcomes of carefully crafted phylogenetic and phylogeographic inference methodologies, the latter of which is able to exploit individual travel histories recorded for infected travellers having entered different European countries.

Protein engineering provides an opportunity to generate new immunogens with desired features. Previously, we have demonstrated that hamster polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) are highly immunogenic and can be employed for the insertion of foreign epitopes at certain surface-exposed positions. In the current study, we have designed pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the target antigen—cellular marker p16INK4A—at its N terminus. Both proteins coexpressed in yeast were self-assembled to pseudotype VLPs harbouring the inserted antigen on the surface. The pseudotype VLPs were used for generation of antibodies against p16INK4Athat represents a potential biomarker for cells transformed by high-risk human papillomavirus (HPV). The pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining of p16INK4Aprotein in malignant cervical tissue. Spleen cells of the immunized mice were used to generate monoclonal antibodies against p16INK4Aprotein. The specificity of antibodies was proven by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value.

Wall-less bacteria known as phytoplasmas are obligate transkingdom parasites and pathogens of plants and insect vectors. These unusual bacteria possess some of the smallest genomes known among pathogenic bacteria, and have never been successfully isolated in artificial culture. Disease symptoms induced by phytoplasmas in infected plants include abnormal growth and often severe yellowing of leaves, but mechanisms involved in phytoplasma parasitism and pathogenicity are little understood. A phage based genomic island (sequence variable mosaic, SVM) in the genome of Malaysian periwinkle yellows (MPY) phytoplasma harbors a gene encoding membrane-targeted proteins, including a putative phospholipase (PL), potentially important in pathogen-host interactions. Since some phytoplasmal disease symptoms could possibly be accounted for, at least in part, by damage and/or degradation of host cell membranes, we hypothesize that the MPY phytoplasma putative PL is an active enzyme. To test this hypothesis, functional analysis of the MPY putative pl gene-encoded protein was carried out in vitro after its expression in bacterial and yeast hosts. The results demonstrated that the heterologously expressed phytoplasmal putative PL is an active lipolytic enzyme and could possibly act as a pathogenicity factor in the plant, and/or insect, host.