Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.

The relationship between biofilm formations and capsule in Haemophilus influenzae.

To evaluate the biofilm formation of non-typeable Haemophilus influenzae (NTHi) and H. influenzae type b (Hib) clinical isolates, we conducted the following study. Serotyping and polymerase chain reaction were performed to identify ß-lactamase-negative ampicillin (ABPC)-susceptible (BLNAS), ß-lactamase-negative ABPC-resistant (BLNAR), TEM-1 type ß-lactamase-producing ABPC-resistant (BLPAR)-NTHi, and Hib. Biofilm formation was investigated by microtiter biofilm assay, as well as visually observation with a scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) in a continuous-flow chamber. As a result, totally 99 strains were investigated, and were classified into 4 groups which were 26 gBLNAS, 22 gBLNAR, 28 gBLPAR-NTHi and 23 Hib strains. The mean OD600 in the microtiter biofilm assay of gBLNAS, gBLNAR, gBLPAR-NTHi, and Hib strains were 0.57, 0.50, 0.34, and 0.08, respectively. NTHi strains were similar in terms of biofilm formations, which were observed by SEM and CLSM. Five Hib strains with the alternated type b cap loci showed significantly increased biofilm production than the other Hib strains. In conclusion, gBLNAS, gBLNAR, and gBLPAR-NTHi strains were more capable to produce biofilms compared to Hib strains. Our data suggested that resistant status may not be a key factor but capsule seemed to play an important role in H. influenzae biofilm formation.

Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.

Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity.

Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.

Salmonella enterotoxin (Stn) regulates membrane composition and integrity.

The mechanism of action of Salmonella enterotoxin (Stn) as a virulence factor in disease is controversial. Studies of Stn have indicated both positive and negative effects on Salmonella virulence. In this study, we attempted to evaluate Stn function and its effects on Salmonella virulence. To investigate Stn function, we first performed in vitro and in vivo analysis using mammalian cells and a murine ileal loop model. In these systems, we did not observe differences in virulence phenotypes between wild-type Salmonella and an stn gene-deleted mutant. We next characterized the phenotypes and molecular properties of the mutant strain under various in vitro conditions. The proteomic profiles of the total cell membrane protein fraction differed between wild type and mutant in that there was an absence of a protein in the mutant strain, which was identified as OmpA. By far-western blotting, OmpA was found to interact directly with Stn. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy, with OmpA localization being analyzed by immunogold labeling. Compared with wild-type Salmonella, the mutant strain had a different pole structure and a thin periplasmic space; OmpA was not seen in the mutant. These results indicate that Stn, via regulation of OmpA membrane localization, functions in the maintenance of membrane composition and integrity.

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes.

Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5'exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that - in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Direct binding of gangliosides to Helicobacter pylori vacuolating cytotoxin (VacA) neutralizes its toxin activity.

Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.

Bactericidal activity in filtrated supernatant of Streptococcus sanguinis against multidrug-resistant Pseudomonas aeruginosa.

In the past decade, multidrug-resistant Pseudomonas aeruginosa (MDRP) infection has become a serious clinical problem, due to the limitation of drug choices to fight against the bacteria. Here we explored the bactericidal activity in the filtrated supernatant of Streptococcus (S.) sanguinis against Pseudomonas (P.) aeruginosa. S. sanguinis is one of the alpha-hemolytic streptococci that commonly reside in the human oral cavity. A strain of S. sanguinis, isolated from the sputum of a pulmonary-disease patient, was cultured for overnight. The filtered supernatant was tested for bactericidal effect using the minimum bactericidal concentration method on 20 strains of P. aeruginosa, including two MDRP and five mucoid-type strains. The viable number of P. aeruginosa was decreased with time after exposing to the filtrated supernatant of S. sanguinis, and collapsed bacteria were detected with electron microscopy. Of the 20 strains, 19 (95%) strains of P. aeruginosa were affected by bactericidal effect. Among other species of bacteria examined, the filtrated supernatant of S. sanguinis showed remarkable bactericidal effect on 49% of indole-positive Proteus species (4/9 strains) and 60% of Acinetobacter (A.) baumannii (6/10 strains). We next investigated the property of bactericidal activity in filtrated supernatant by treating with proteinase K or autoclave. There was no change in the bactericidal activity of the filtrated supernatant after each treatment, excluding the involvement of protein and plasmid. Here, we identify the bactericidal activity in the filtrated supernatant of S. sanguinis against MDRP. This unexpected observation may contribute to the development of a novel therapeutic drug against P. aeruginosa.

Variation in the attachment of Streptococcus pneumoniae to human pharyngeal epithelial cells after treatment with S-carboxymethylcysteine.

S-carboxymethylcysteine (S-CMC) is a mucolytic agent that can prevent respiratory infection by decreasing the attachment of respiratory pathogens to human pharyngeal epithelial cells (HPECs). Streptococcus pneumoniae is a major cause of respiratory infections. A previous study revealed that treatment of S. pneumoniae with S-CMC caused a decrease in the attachment of this bacterium to HPECs. In the present study we found that the effect of S-CMC varied according to hosts and strains. S-CMC treatment altered the surface structure of S. pneumoniae, resulting in a decrease of attachment, without affecting the virulence of the bacteria.

Trypanocidal activity of extracts and compounds from the stem bark of Anogeissus leiocarpus and Terminalia avicennoides.

The antitrypanosomal activity of methanolic extracts of Anogeissus leiocarpus and Terminalia avicennoides were evaluated in vitro against four strains of Trypanosoma species with minimum inhibitory concentration (MIC) value range of 12.5-50 mg/ml. Successive fractionations of the two plant extracts in water, butanol and ethyl acetate gave a range of activity (MIC, 20 to > or =50 microg/ml). Activity-guided and chromatographic analysis of butanolic fractions on Sephadex LH-20 column followed by high-performance liquid chromatography, nuclear magnetic resonance analysis and both ultraviolet and thin layer chromatography revealed hydrolysable tannins with a range of activity (MIC, 7.5-27.5 microg/ml or 14-91 microM). Effect of the compounds on fibroblasts did not reveal serious toxicity at moderate concentration but is concentration dependent.

Temporary shift of microfilariae of Brugia pahangi from the lungs to muscles in Mongolian jirds, Meriones unguiculatus, after a single injection of diethylcarbamazine.

A single-dose treatment with diethylcarbamazine (DEC) reduced microfilaria (mf) counts of Brugia pahangi by >90% at 30 min post-treatment in Mongolian jirds (Meriones unguiculatus). The reduction was followed by a rapid increase in microfilaremia, with the count reaching pretreatment level in 3 hr. The mechanisms behind this temporary reduction of mf were investigated. Without treatment, mf accumulated in the lungs. At 30 min post-treatment, they had moved from the lungs and accumulated in the muscle. At the same time, electron microscopy revealed many mf in the muscle interstitium. DEC concentrations at 30 min were much lower in the muscle (12.2 microg/g of tissue) than in the lungs, liver, and kidneys (19.8-40.7 microg/g), all of which declined to < 0.6 microg/g by 3 hr. The presence of mf in the muscle would be advantageous for avoiding high DEC concentrations, and their extravascular location could prevent attack by host effector cells.

Role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells.

The goal of this study was to determine the role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells. Strain 2951 and its P(k) mutant strain 2951 galE were used in this study. This study suggests that the P(k) epitope of LOS is not an adhesin for M. catarrhalis, but plays a crucial role by its surface charge in the initial stage of attachment.

In vitro cultivation and electron microscopy characterization of Trachipleistophora anthropophthera isolated from the cornea of an AIDS patient.

We describe an in vitro culture technique for a microsporidian isolated from the corneal biopsy of an HIV-infected patient. The corneal biopsy was inoculated into a monolayer culture of fibroblasts derived from newborn mouse brain and incubated at 37 degrees C in an atmosphere of 5% CO2. Minimum essential medium supplemented with 2% fetal bovine serum appeared to be an optimum medium for growth and maintenance of the parasite and for production of large numbers of spores. This microsporidian was identified as Trachipleistophora anthropophthera based on ultrastructural features. It forms two types of sporophorous vesicles and two types of spores simultaneously: polysporous vesicle type I with eight or more oval spores, 3.7-4.0 microm by 2.0-2.3 microm, and bisporous vesicle type II with two round spores, 1.7-2.2 microm by 1.6-2.0 microm in size.

Fusion PCR generated Japanese encephalitis virus/dengue 4 virus chimera exhibits lack of neuroinvasiveness, attenuated neurovirulence, and a dual-flavi immune response in mice.

The first flavivirus chimera encoding dengue 4 virus (D4) PrM and E structural proteins in a Japanese encephalitis virus (JEV) backbone was successfully generated using the long-PCR based cDNA-fragment stitching (LPCRcFS) technique, demonstrating the technique's applicability for rapid preparation of flavivirus chimeras. The JEV/D4 chimera multiplied at levels equal to JEV and D4 in the mosquito cell line C6/36, while in a mouse neuronal cell line (N2a) JEV replicated efficiently, but JEV/D4 and D4 did not. In mouse challenge experiments, JEV/D4 showed a lack of neuroinvasiveness similar to D4 when inoculated intraperitoneally, but demonstrated attenuated neurovirulence (LD50=3.17 x 10(4) f.f.u.) when inoculated intracranially. It was also noted that mice receiving intraperitoneal challenge with JEV/D4 possessed D4-specific neutralization antibody and in addition clearly showed resistance to JEV intraperitoneal challenge (at 100 x LD50). This suggests that immunity to anti-JEV non-structural protein(s) offers protection against JEV infection in vivo. Dengue secondary infection was also simulated by challenging mice pre-immunized with dengue 2 virus, with D4 or JEV/D4. Mice showed higher secondary antibody response to challenge with JEV/D4 than to D4, at 210,000 and 37,000 averaged ELISA units, respectively. Taken together, aside from demonstrating the LPCRcFS technique, it could be concluded that the PrM and E proteins are the major determinant of neuroinvasiveness for JEV. It is also expected that the JEV/D4 chimera with its pathogenicity in mice and atypical immune profile, could have applications in dengue prophylactic research, in vivo efficacy assessment of dengue vaccines and development of animal research on models of dengue secondary infection.

Intracellular visualization of tetrodotoxin (TTX) in the skin of a puffer Tetraodon nigroviridis by immunoenzymatic technique.

Micro-distributions of tetrodotoxin (TTX) in the skin of a brackish-water puffer Tetraodon nigroviridis were investigated by means of a monoclonal anti-TTX antibody under light and transmission electron microscope. In light microscopy TTX antigen was visualized as brown color in undifferentiated basal cells and succiform cells of the skin, while in electron microscopy TTX was detected as black dots of immunogold in lysosomes of basal cells. From the results, it can be inferred that when TTX from the blood plasma of T. nigroviridis enters into the undifferentiated basal cells through diffusion, it is taken to the lysosomes by phagocytosis and exists there through binding with internal constituent(s) of the organelles.

Acid aspiration induces bacterial pneumonia by enhanced bacterial adherence in mice.

The issue of whether acid aspiration facilitates bacterial pneumonia caused by Pseudomonas aeruginosa by enhanced bacterial adherence was examined in mice. Survival or the number of bacteria in lung tissues was evaluated after an intratracheal challenge of hydrochloric acid (HCl), a sublethal dose of P. aeruginosa, or both in mice. Bacterial adherence to the tracheal epithelium after acid aspiration was also examined by scanning electron microscopy. A simultaneous intratracheal challenge of 50 microl of 10(-1) N HCl, but not 10(-2) to 10(-4) N HCl, combined with a sublethal dose of P. aeruginosa significantly increased the number of bacteria in the lung tissues and decreased survival, while all mice that received either HCl or P. aeruginosa survived. Significantly higher numbers of adherent bacteria on the tracheal epithelium were found in mice that received 10(-1)N HCl, compared with mice that received HCl (10(-2) to 10(-4) N) or saline. These data indicate that acid aspiration induced airway epithelial injury and enhanced P. aeruginosa adherence to the epithelium, and led to the subsequent development of bacterial pneumonia in mice. Enhanced bacterial adherence on the acid-injured epithelium may explain fatal bacterial pneumonias in patients with respiratory aspiration of gastric contents.