The role of the DE and EF loop of BKPyV VP1 in the serological cross-reactivity between subtypes.

BK virus (BKPyV) is a causative agent of BKPyV-associated nephropathy and graft rejections in kidney transplant patients. It establishes persistent infection in the kidneys, which can lead to reactivation in an immunosuppressed state or transmission to kidney recipients. Complications in the case of donor-derived infections can be caused by differences between the four known BKPyV subtypes, as prior infection with one subtype does not guarantee protection against de novo infection with other subtypes. The recipient and donor pretransplant serotyping is not routinely performed since simple ELISA tests employing antigens derived from the major viral capsid protein 1 (VP1) are hindered by the high cross-reactivity of anti-VP1 antibodies against all subtypes. Identifying subtype-specific epitopes in VP1 could lead to the design of specific antigens and the improvement of serodiagnostics for kidney transplantation. We aimed to study the surface residues responsible for the interactions with the subtype-specific antibodies by focusing on the DE and EF loops of VP1, which have only a small number of distinct amino acid differences between the most common subtypes, BKPyV-I and BKPyV-IV. We designed two mutant virus-like particles (VLPs): we introduced BKPyV-I characteristic amino acid residues (either H139N in the DE loop or D175E and I178V changes in the EF loop) into the base sequence of a BKPyV-IV VP1. This way, we created BKPyV-IV mutant VLPs with the sequence of either the BKPyV-I DE loop or the BKPyV-I EF loop. These mutants were then used as competing antigens in an antigen competition assay with a panel of patient sera, and changes in antibody reactivity were assessed by ELISA. We found that the changes introduced into the BKPyV-IV VP1 EF loop restrict antibody recognition in most samples and that converting the BKPyV-IV DE loop into its BKPyV-I equivalent attracts anti-VP1 BKPyV-I antibodies. Although our results did not lead to the discovery of a subtype-specific epitope on the VP1, they suggested that the arrangement of the EF loop in VP1 might dictate the mode of interaction between virus and anti-VP1 antibodies in general and that the interactions between the antibodies and the viral capsid might be very complex. Consequently, an antigen competition assay as an assay to distinguish between BKPyV serotypes might prove difficult to interpret.

VirPorters: Insights into the action of cationic and histidine-rich cell-penetrating peptides.

The utilization of nanoparticles for the intracellular delivery of theranostic agents faces one substantial limitation. Sequestration in intracellular vesicles prevents them from reaching the desired location in the cytoplasm or nucleus to deliver their cargo. We investigated whether three different cell-penetrating peptides (CPPs), namely, octa-arginine R8, polyhistidine KH27K and histidine-rich LAH4, could promote cytosolic and/or nuclear transfer of unique model nanoparticles-pseudovirions derived from murine polyomavirus. Two types of CPP-modified pseudovirions that carry the luciferase reporter gene were created: VirPorters-IN with CPPs genetically attached to the capsid interior and VirPorters-EX with CPPs noncovalently associated with the capsid exterior. We tested their transduction ability by luciferase assay and monitored their presence in subcellular fractions. Our results confirmed the overall effect of CPPs on the intracellular destination of the particles and suggested that KH27K has the potential to improve the cytosolic release of pseudovirions. None of the VirPorters caused endomembrane damage detectable by the Galectin-3 assay. Remarkably, a noncovalent modification was required to promote high transduction of the reporter gene and cytosolic delivery of pseudovirions mediated by LAH4. Together, CPPs in different arrangements have demonstrated their potential to improve pseudovirion invasion into cells, and these findings could be useful for the development of other nanoparticle-based delivery systems.

Cell-penetrating peptides in the intracellular delivery of viral nanoparticles.

Cell-penetrating peptides (CPPs) are a promising tool for the intracellular delivery of cargo. Due to their ability to cross membranes while also cotransporting various cargoes, they offer great potential for biomedical applications. Several CPPs have been derived from viral proteins with natural roles in the viral replication cycle that require them to breach or fuse to cellular membranes. Additionally, the ability of viruses to cross membranes makes viruses and virus-based particles a convenient model for research on nanoparticle delivery and nanoparticle-mediated gene therapy. In this chapter, we aim to characterize CPPs derived from both structural and nonstructural viral proteins. Their function as enhancers of viral infection and transduction by viral nanoparticles as well as the main features of viral CPPs employed in intracellular cargo delivery are summarized to emphasize their potential use in nanomedicine.

A polyomavirus detected in American black bear (Ursus americanus).

Polyomaviruses are ancient DNA viruses that infect several species of animals. While recognition of the family Polyomaviridae has grown rapidly, there are few studies that consider their potential association with disease. Carnivora are a diverse and widespread order affected by polyomaviruses (PyVs) that have co-evolved with their hosts for millions of years. PyVs have been identified in sea lions, raccoons, badgers, Weddell seals, and dogs. We have discovered a polyomavirus, tentatively named "Ursus americanus polyomavirus 1" (UaPyV1) in black bears (Ursus americanus). UaPyV1 was detectable in various tissues of six out of seven bears submitted for necropsy. Based on viral phylogenetic clustering and detection of the virus in multiple individuals, we suggest that black bears are the natural hosts for UaPyV1. In this albeit small group, there is no clear relationship between UaPyV1 infection and any specific disease.

The Protein Corona Does Not Influence Receptor-Mediated Targeting of Virus-like Particles.

Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.

Prevalence and Risk Factors for Oral HPV in Healthy Population, in Central Europe.

Background/Aim: The incidence of oropharyngeal tumours induced by human papillomaviruses (HPV) is ever increasing. Information about oral HPV prevalence and its risk factors are very important for future screening and early diagnosis of the disease. The present study aimed to assess oral HPV prevalence in healthy population and risk factors for HPV infection, since this data is scarce or even missing in Central Europe. Patients and Methods: HPV prevalence in oral rinse and HPV-specific antibodies in peripheral blood were investigated in two groups of healthy participants. Group I consisted of 294 students who reached sexual maturity after the HPV vaccine had been licensed with mean age 23.2 years, and Group II of 215 unvaccinated participants with the mean age 55.7 years. Additionally, the risk factors were evaluated. Results: In Group I, 2% of participants were positive for oral HPV DNA. A statistically significantly higher rate (8.8%) was found in Group II. The seropositivity rates for anamnestic HPV antibodies were comparable in both groups. None of the analysed risk factors was significantly associated with oral HPV positivity. Conclusion: The lower prevalence of oral HPV DNA in younger participants suggests the positive influence of vaccination against oral HPV.

Influence of cell-penetrating peptides on the activity and stability of virus-based nanoparticles.

Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.

The Utilization of Cell-Penetrating Peptides in the Intracellular Delivery of Viral Nanoparticles.

Viral particles (VPs) have evolved so as to efficiently enter target cells and to deliver their genetic material. The current state of knowledge allows us to use VPs in the field of biomedicine as nanoparticles that are safe, easy to manipulate, inherently biocompatible, biodegradable, and capable of transporting various cargoes into specific cells. Despite the fact that these virus-based nanoparticles constitute the most common vectors used in clinical practice, the need remains for further improvement in this area. The aim of this review is to discuss the potential for enhancing the efficiency and versatility of VPs via their functionalization with cell-penetrating peptides (CPPs), short peptides that are able to translocate across cellular membranes and to transport various substances with them. The review provides and describes various examples of and means of exploitation of CPPs in order to enhance the delivery of VPs into permissive cells and/or to allow them to enter a broad range of cell types. Moreover, it is possible that CPPs are capable of changing the immunogenic properties of VPs, which could lead to an improvement in their clinical application. The review also discusses strategies aimed at the modification of VPs by CPPs so as to create a useful cargo delivery tool.

Prevalence of antibodies against BKPyV subtype I and IV in kidney transplant recipients and in the general Czech population.

Active infection with BK polyomavirus (BKPyV) may cause serious complications in transplantation settings. Recently, the level of BKPyV IgG seroreactivity in graft donors has been shown to predict viremia and BKPyV-associated nephropathy in kidney transplant (KTx) recipients. Pretransplantation testing of the donor and recipient BKPyV serostatus could, therefore, identify patients at high risk. For the development of serological immunoassays, antibody response to the predominant BKPyV subtypes (BKPyV-I and BKPyV-IV) was studied using virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). VLPs made from the capsid protein, VP1, derived from BKPyV-I and BKPyV-IV subtypes were produced using a baculovirus expression system and used as antigens. The tests were used for IgG antibody determination in 50 KTx recipients and 111 healthy blood donors. While 87% of samples reacted with mixed BKPyV-I and BKPyV-IV antigens, only 49% of samples were reactive in both ELISA tests when using BKPyV-I or BKPyV-IV antigens separately. Twenty-seven percent of healthy blood donors and 26% of KTx recipients were reactive only with BKPyV-I, while 9% and 20% were reactive only with BKPyV-IV, respectively. To determine the specificities of the antigens, selected seropositive samples were retested after preadsorption with soluble BKPyV-I, BKPyV-IV, or JC polyomavirus antigens. The experiments confirmed that recombinant VP1 VLP-based ELISAs predominantly detected BKPyV type-specific antibodies. The results imply that anti-BKPyV antibody ELISA tests should contain a mixture of subtype-specific VLP-based antigens instead of antigen derived from the most prevalent BKPyV-I subtype. The tests can be used for serological surveys of BKPyV infection and improved KTx patient management.