The pathogenesis of 3 neurotropic flaviviruses in a mouse model depends on the route of neuroinvasion after viremia.

Neurotropic flavivirus infection of humans results in viremia subsequently; in some cases, it causes meningitis encephalomyelitis, although the pathways from viremia to central nervous system (CNS) invasion are uncertain. Here, we intravenously infected BALB/c mice with 3 neurotropic flaviviruses, then examined the clinical manifestations and histopathologic changes. The Sofjin strain of tick-borne encephalitis virus-infected mice exhibited dose-dependent survival.

Poly-γ-glutamic acid nanoparticles and aluminum adjuvant used as an adjuvant with a single dose of Japanese encephalitis virus-like particles provide effective protection from Japanese encephalitis virus.

To maintain immunity against Japanese encephalitis virus (JEV), a formalin-inactivated Japanese encephalitis (JE) vaccine should be administered several times. The repeated vaccination is not helpful in the case of a sudden outbreak of JEV or when urgent travel to a high-JEV-risk region is required; however, there are few single-injection JE vaccine options. In the present study, we investigated the efficacy of a single dose of a new effective JE virus-like particle preparation containing the JE envelope protein (JE-VLP).

Purification and concentration of non-infectious West Nile virus-like particles and infectious virions using a pseudo-affinity Cellufine Sulfate column.

Affinity column chromatography is a promising method for the purification of flavivirus particles that can supplement or potentially replace diafiltration and sucrose density centrifugation. In this study, the purification of West Nile Virus (WNV) antigens via Cellufine Sulfate column chromatography was examined. Virus-like particles (VLPs) produced by the expression of the prM and E genes were separated from most of the contaminant proteins with 0.

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate.

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients.

Virus detection using Viro-Adembeads, a rapid capture system for viruses, and plaque assay in intentionally virus-contaminated beverages.

Intentional contamination of beverages with microbes is one type of bioterrorist threat. While bacteria and fungus can be easily collected by a centrifuge, viruses are difficult to collect from virus-contaminated beverages. In this study, we demonstrated that Viro-Adembeads, a rapid-capture system for viruses using anionic polymer-coated magnetic beads, collected viruses from beverages contaminated intentionally with vaccinia virus and human herpesvirus 8.

Covalent bonded Gag multimers in human immunodeficiency virus type-1 particles.

The oligomerization of HIV-1 Gag and Gag-Pol proteins, which are assembled at the plasma membrane, leads to viral budding. The budding generally places the viral components under non-reducing conditions. Here the effects of non-reducing conditions on Gag structures and viral RNA protection were examined.

Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus.

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain.

Expression and characterization of bovine lactoperoxidase by recombinant vaccinia virus.

Lactoperoxidase (LPO) is a 78 kDa heme-containing oxidation-reduction enzyme present in milk, found in physiological fluids of mammals. LPO has an antimicrobial activity, and presumably contribute to the protective functions of milk against infectious diseases. In this study, recombinant vaccinia virus expressing bovine LPO (vv/bLPO) was constructed.

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition.

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately.

An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection.

The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood.

Amino acid 36 in the human immunodeficiency virus type 1 gp41 ectodomain controls fusogenic activity: implications for the molecular mechanism of viral escape from a fusion inhibitor.

We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3.

Japanese encephalitis subunit vaccine composed of virus-like envelope antigen particles purified from serum-free medium of a high-producer J12#26 cell clone.

A stable cell clone, J12#26, which continuously secretes large amounts of the envelope (E) antigen of Japanese encephalitis (JE) virus (J. Virol. 77 (2003) 8745) was adapted to serum-free medium.

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry.

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated.

Nucleolin and the packaging signal, psi, promote the budding of human immunodeficiency virus type-1 (HIV-1).

Gag proteins of human immunodeficiency virus type 1 (HIV-1) play a pivotal role in the budding of the virion, in which the zinc finger motifs of the gag proteins recognize the packaging signal of genomic RNA. Nucleolin, an RNA-binding protein, is identified as a cellular protein that binds to murine leukemia virus (MuLV) gag proteins and regulates the viral budding, suggesting that HIV-1 gag proteins, the packaging signal, psi and nucleolin affect the budding of HIV-1. Here we report that nucleolin enhances the release of HIV-1 virions which contain psi.

Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection.

Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the gag gene of six field isolates by adopting an env/nef-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SLFNTVAVL and SVYNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KYRLKHLVW, QYRLKHIVW, and RYRLKHLVW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells.

Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine.

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.

Processing of the HTLV-II envelope precursor glycoprotein gp63 by furin is essential for cell fusion activity.

To investigate the relationship between the fusogenic properties of HTLV-II and the processing of the envelope precursor glycoprotein gp63, recombinant cowpox virus expressing this protein was used to infect a range of cell lines derived from different species. Syncytium formation and gp63 processing were observed in all cells with the exception of LoVo cells, which are known to have a dysfunctional form of the endoprotease, furin. Furin has been shown to be necessary for the processing of a number of viral envelope glycoproteins, and gp63 contains a consensus sequence (305)Arg-Arg-Arg-Arg, which is a furin substrate motif.

Role of nucleotide sequences in the V3 region in efficient replication of CCR5-utilizing human immunodeficiency virus type 1 in macrophages.

Macrophages express both CXCR4 and CCR5 coreceptors, but restrict X4 HIV-1 replication unless the Env-V3 region, a major determinant of cell tropism, is exchanged with that of R5 HIV-1. As the V3 exchange concomitantly alters the nucleotide sequences, we introduced silent mutations in the V3 or C2 region of macrophage-tropic R5 JRFL without changing the amino acids. Immunoblot analysis confirmed that viral proteins including Env-gp120 were similarly incorporated in wild-type (wt) and mutant virions.

An immunodominant neutralization epitope on the 'thumb' subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies.

An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated.