Graphene-Assisted Synthesis of 2D Polyglycerols as Innovative Platforms for Multivalent Virus Interactions.

2D nanomaterials have garnered widespread attention in biomedicine and bioengineering due to their unique physicochemical properties. However, poor functionality, low solubility, intrinsic toxicity, and nonspecific interactions at biointerfaces have hampered their application in vivo. Here, biocompatible polyglycerol units are crosslinked in two dimensions using a graphene-assisted strategy leading to highly functional and water-soluble polyglycerols nanosheets with 263 ± 53 nm and 2.7 ± 0.2 nm average lateral size and thickness, respectively. A single-layer hyperbranched polyglycerol containing azide functional groups is covalently conjugated to the surface of a functional graphene template through pH-sensitive linkers. Then, lateral crosslinking of polyglycerol units is carried out by loading tripropargylamine on the surface of graphene followed by lifting off this reagent for an on-face click reaction. Subsequently, the polyglycerol nanosheets are detached from the surface of graphene by slight acidification and centrifugation and is sulfated to mimic heparin sulfate proteoglycans. To highlight the impact of the two-dimensionality of the synthesized polyglycerol sulfate nanosheets at nanobiointerfaces, their efficiency with respect to herpes simplex virus type 1 and severe acute respiratory syndrome corona virus 2 inhibition is compared to their 3D nanogel analogs. Four times stronger in virus inhibition suggests that 2D polyglycerols are superior to their current 3D counterparts.

Targeted Alteration of Antibody-Based Immunodominance Enhances the Heterosubtypic Immunity of an Experimental PCV2 Vaccine.

Despite the availability of commercial vaccines which can effectively prevent clinical signs, porcine circovirus type 2 (PCV2) continues to remain an economically important swine virus, as strain drift, followed by displacement of new subtypes, occurs periodically. We had previously determined that the early antibody responses to the PCV2 capsid protein in infected pigs map to immunodominant but non-protective, linear B cell epitopes. In this study, two of the previously identified immunodominant epitopes were mutated in the backbone of a PCV2b infectious clone, to rationally restructure the immunogenic capsid protein. The rescued virus was used to immunize 3-week-old weanling piglets, followed by challenge with a virulent heterologous PCV2d strain. As expected, immunodominant antibody responses to the targeted epitopes were abrogated in vaccinated pigs, while a broadening of the virus neutralization responses was detected. Vaccinated pigs were completely protected against challenge viral replication, had reduced microscopic lesions in lymphoid organs and gained significantly more body weight when compared to unvaccinated pigs. Thus, the experimental PCV2 vaccine developed was highly effective against challenge, and, if adopted commercially, can potentially slow down or eliminate new strain creation.

Equine Alphaherpesviruses Require Activation of the Small GTPases Rac1 and Cdc42 for Intracellular Transport.

Viruses utilize host cell signaling to facilitate productive infection. Equine herpesvirus type 1 (EHV-1) has been shown to activate Ca2+ release and phospholipase C upon contact with α4ß1 integrins on the cell surface. Signaling molecules, including small GTPases, have been shown to be activated downstream of Ca2+ release, and modulate virus entry, membrane remodeling and intracellular transport. In this study, we show that EHV-1 activates the small GTPases Rac1 and Cdc42 during infection. The activation of Rac1 and Cdc42 is necessary for virus-induced acetylation of tubulin, effective viral transport to the nucleus, and cell-to-cell spread. We also show that inhibitors of Rac1 and Cdc42 did not block virus entry, but inhibited overall virus infection. The Rac1 and Cdc42 signaling is presumably orthogonal to Ca2+ release, since Rac1 and Cdc42 inhibitors affected the infection of both EHV-1 and EHV-4, which do not bind to integrins.

Differentially-Charged Liposomes Interact with Alphaherpesviruses and Interfere with Virus Entry.

Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. Here, we investigated the interaction of differently charged phospholipids with virus particles and determined their influence on infection. Our data show that liposomes containing negatively charged PS or positively charged DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium) inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while, presumably, acting through different mechanisms. Charged phospholipids were found to have antiviral effects and may be used to inhibit EHV-1 infection.

Efficacy of a Commercial PCV2a Vaccine with a Two-Dose Regimen Against PCV2d.

Porcine circovirus type 2, the causative agent of porcine circovirus associated diseases (PCVAD), consists of three major genotypes PCV2a, 2b and 2d. Current commercial vaccines contain the first-identified PCV2a's capsid protein or whole virions. Outbreaks of PCVAD, caused by the recently identified PCV2d in vaccinated herds have raised concerns regarding the efficacy of current PCV2a vaccines against PCV2d. Thus, the primary objective of this study was to assess the efficacy of a two-dose regimen for the recently reformulated Fostera PCV MetaStim vaccine, to determine if reformulation with the squalene oil adjuvant and two-dose regimen improves the threshold of protection enough to eliminate viremia in a vaccination and challenge model. Two groups of seven pigs each were vaccinated with the commercial vaccine or PBS, and challenged with the PCV2d virus. Strong pre-challenge virus neutralizing responses were detected against all three genotypes. Post-challenge viremia was not completely eliminated as expected but a 2 log10 mean reduction in viral load was achieved in vaccinated pigs. Vaccinated pigs had a mean score of 0 for pathological evaluation, while unvaccinated pigs had a score of 6.6. In conclusion, the reformulated Fostera PCV MetaStim PCV2a-based vaccine provided significant heterologous protection and was effective against PCV2d.

Identification of heterologous Torque Teno Viruses in humans and swine.

Torque Teno Viruses (TTVs) are ubiquitous viruses which are highly prevalent in several mammalian species. Human TTV's are epidemiologically associated with several human disease conditions such as respiratory illnesses, auto-immune disorders and hepatitis. Recently it was found that swine TTV's (TTSuVs) can act as primary pathogens. The common occurrence of TTVs as environmental contaminants and the increasing interest in the use of swine organs for xenotransplantation lend importance to the question of whether TTV's can cross-infect across species. In this study, we examined human and swine sera by swine or human TTV-specific PCRs, to determine whether swine TTVs (TTSuV) DNA can be detected in humans and vice versa. Surprisingly, both human and TTSuV DNA were present in a majority of the samples tested. Transfection of human PBMC's with TTSuV1 genomic DNA resulted in productive viral infection which was sustained for the three serial passages tested. Lymphoproliferative responses in infected human PBMCs were diminished when compared to the controls. Furthermore, mild to moderate antibody responses against the TTSuV1 ORF2 protein was detected in 16 of the 40 human sera by ELISA. Therefore, these study findings provide initial and fundamental evidence for possible cross-species transmission of TTVs.

Antigenic Determinants of Possible Vaccine Escape by Porcine Circovirus Subtype 2b Viruses.

Currently available commercial vaccines against porcine circovirus strain 2 (PCV2) solely target the PCV2a genotype. While PCV2 vaccines are highly effective in preventing clinical signs, PCV2b has dominated over the PCV2a genotype in prevalence, corresponding with the introduction of PCV2a vaccines. A recently emerged PCV2b recombinant with an additional amino acid in the capsid protein, designated the mutant PCV2b (mPCV2b), is cause for concern due to its increased virulence and rapid spread. The accumulation of recent evidence for the increased genetic diversity in PCV2 suggests that current vaccines against PCV2a may be inducing selection pressure and driving viral evolution. In this study, the hypothesis that differences in key immune epitopes between the PCV2a vaccine strains, a classical PCV2b strain called PCV2b 41513 obtained from a vaccine-failure case, and mPCV2b strains could promote vaccine escape was tested using immuno-informatic tools. In the major viral proteins, 9 of the 18 predicted swine leukocyte antigens (SLA) class-I epitopes, 8 of the 22 predicted SLA class-II epitopes, and 7 of the 25 predicted B cell epitopes varied between the vaccine and field strains. A majority of the substitutions in both the T- and B-cell epitopes were located in the capsid protein. Some B- and T-cell epitopes that were identified as immunogenic in the vaccine strain were not identified as epitopes in the field strains, indicating a subtle shift in the antigenic profile of the field strains. Several nonconserved epitopes had both predicted B- and T-cell functions. Therefore, substitutions in the dual epitopes could affect both arms of the immune response simultaneously, causing immune escape. Our findings support further rational design of PCV2 vaccines to increase the current threshold of protection.