Quantification of virus-like particles suggests viral infection in corals affected by Porites tissue loss

Porites tissue loss is a common disease of Porites compressa on Hawaiian reefs. Despite its prevalence, to date, the aetiological agent of the disease has not been found. The apparent lack of a microbial causative agent in the similar disease Porites bleaching with tissue loss, as well as increasing evidence of viral infections in scleractinian corals and Symbiodinium, led us to hypothesise that a virus may be responsible. Electron microscopy revealed the presence of numerous and varied virus-like particles (VLPs) in healthy and diseased P. compressa colonies. While overall virus numbers were similar in all samples, the abundance of a group of icosahedral VLPs differed significantly between healthy and diseased colonies. While not conclusive, these results suggest that viruses may play a role in this disease, and provide a basis for further studies.     

Comparison of the sensitivity of the serological latex and ELISA tests

A sensitivity of the serological latex and ELISA tests were compared in carnation mottle virus diagnosis. For the latex test carnation mottle virus (CaMV) antiserum was sensibilized with latex suspension for RF-test. Sensibilized antiserum was used in 1: 200 dilution, as compared with fresh antiserum. For ELISA the γ-globuline fraction of antiserum was conjugated with alkaline phosphatase. The optimal dilution in both, CaMV fraction of antisera for coating of plates and γ-globuline-enzyme conjugate were in the ratio of 1: 500, 2 μg of antibodies in 1 ml. The dilution end point of carnation mottle virus in sap from carnation leaves was 1.6 × 10^?4 to 1.25 × l0^?5 and 1 × 10^?4 to 1.25 × l0^?5, when serological latex and ELISA tests were used. As indicated, ELISA as compared to the latex test was found to be more sensitive for carnation mottle diagnosis. As the latex test is considered to be simpler and cheaper, and in addition, showing the same assurance as the biological test onChenopodium amaranticolor, the latex test is recommended for carnation mottle virus detection.     

Ultrastructure of Inclusions Formed by Ribgrass Mosaic Virus in Leaf Cells

Diffrent types of cytoplasmic inclusions were observed in ultrathin sections of plants systemically infected with three different strains of ribgrass mosaic virus (RMV) (tobamovirus group). Tissue from uninoculated plants did not contain such inclusions. Most common were “rounded plates” consisting of layers of aligned virus particles 300 nm long. RMV also induced angled layer aggregates in Capsicum annuum plants. A novel type of inclusion for the tobamovirus group were the abundant spiral aggregates found in Digitalis purpurea, systemically infected with strain D of RMV. In these aggregates the virions become circularly arranged around a center. The orientation of the particles changes in such a way that virions being 300 nm apartare cut in the longitudinal and in the transverse direction respectively.     

The Mechanical transmission and some properties of a virus disease of cow-parsnip,Heracleum sphondylium L

The described virus of cow-parsnip,Heracleum sphondylium L., was found in three ruderal localities of Greater Praha. The symptoms are manifested by decolorations which consist of bright yellow areas spreading from the centre of the leaf blade along the main veins. These symptoms appear severely in May. Under higher temperatures and in a chronic stage of infection the symptoms are more or less masked. The disease is mechanically transmissible to parsley, coriander, parsnip, dill, sowbane,Chenopodium quinoa and C.giganteum. The author failed to transmit the disease to celery, carrot, caraway and to 27 species of differential host plants, he failed in the transmission of the virus by the dodder,Cuscuta campestris YUNCK., too. Thermal inactivation point of the virus lies between 51° and 55° C. Infectivity of extracted sap was lost after 2 days at room temperature.     

Biological control of the oil palm pestLatoia viridissima [Lepidoptera,limacodidae], in Cote d'Ivoire,by a new picornavirus

Among the major oil palm pest insects in the Côte d'Ivoire,Latoia viridissima Holland [Lepidoptera, Limacodidae] is the most frequently observed defoliator. During a pullulation of this species, a natural epizootic permitted us to demonstrate the occurrence of a small isometric RNA virus of 30nm in diameter. The buoyant density of the virus particles was 1.34. The virus capsid contained 2 major proteins with molecular weights of 30,000 (55%) and 31,000 (20%) and 3 minor proteins. One genome component was detected with molecular weight 2,9×10⁶. Agarose gel diffusion tests showed this virus was distinct from any other described insect Picornavirus. Trials with different doses of viral suspensions were tested on industrial oil palm plantation, allocated byL. viridissima, from ground level, using an automatic air carried sprayer. One week after the treatment, a mortality gradient, increasing from 11 to 61% according to the dose applied, was obtained. Two weeks after the treatment the mortality reached 92% of the larvae in the treated parcels. During the next generation, the number of caterpillars on the same parcel was very low.     

Vectoring gypsy moth nuclear polyhedrosis virus byApanteles melanoscelus [Hym.: Braconidae]

Gypsy mothLymantria dispar L. larvae were exposed toApanteles melanoscelus (Ratzeburg) females contaminated with nuclear polyhedrosis virus. Three methods of contamination (ovipositor, total body surface, and exposure to infected hosts) and two exposure periods (2 and 24 hours) were tested. A significantly greater incidence of larval mortality caused by virus occurred among larvae exposed to contaminated than among larvae exposed to uncontaminated parasites for 2 and 24 hours. No significant differences occurred in larval mortality caused by virus for the 3 methods of contamination for the 2- and 24-hour tests or in parasite emergence from larvae parasitized by contaminated or uncontaminated parasites.     

The pathogenicity of an entomopoxvirus fromOthnonius batesi [Col.: Scarabaeidae] and its possible use as a control agent

In a natural population ofOthnonius batesi Oll. at Glen Innes, N.S.W., 4.0% of larvae in 1972 and 2.2% in 1973 were exhibiting symptoms of virus infection, whilst 0.8% of pupae and adults from the same population were infected in 1972. These figures, and field observations of infected larvae, suggested that the pathogenicity of the virus was low. In laboratory experiments withO. batesi the infection had little effect on mortality, and no significant effect on duration of the first instar, food intake, or larval growth. The vast accumulation of virus in the fat body probably results in mortality prior to, or during, the pupal period. Temperature had a marked effect on both % infection (optimum 30°C) and production of viral spheroids (optimum 25°C). Very little viral development occurred below 20°C. In a host range study onlyO. batesi, Rhopaea verreauxi Blanch. andR. morbillosa Blkb. were infectedper os. The possible use of the virus as a control agent is discussed.     

Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579

Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.     

Anti-influenza effect of Cordyceps militaris through immunomodulation in a DBA/2 mouse model

The immune-modulatory as well as anti-influenza effects of Cordyceps extract were investigated using a DBA/2 mouse model. Three different concentrations of Cordyceps extract, red ginseng extract, or drinking water were orally administered to mice for seven days, and then the mice were intranasally infected with 2009 pandemic influenza H1N1 virus. Body weight changes and survival rate were measured daily post-infection. Plasma IL-12, TNF-α, and the frequency of natural killer (NK) cells were measured on day 4 post-infection. The DBA/2 strain was highly susceptible to H1N1 virus infection. We also found that Cordyceps extract had an antiinfluenza effect that was associated with stable body weight and reduced mortality. The anti-viral effect of Cordyceps extract on influenza infection was mediated presumably by increased IL-12 expression and greater number of NK cells. However, high TNF-α expression after infection of H1N1 virus in mice not receiving treatment with Cordyceps extract suggested a two-sided effect of the extract on host immune regulation.     

Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system

Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg?·?g^?1 leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.     

Fluorescence imaging of microbe-containing particles shot from a two-stage Light-gas gun into an aerogel

We have proposed an experiment (the Tanpopo mission) to capture microbes on the Japan Experimental Module of the International Space Station. An ultra low-density silica aerogel will be exposed to space for more than 1 year. After retrieving the aerogel, particle tracks and particles found in it will be visualized by fluorescence microscopy after staining it with a DNA-specific fluorescence dye. In preparation for this study, we simulated particle trapping in an aerogel so that methods could be developed to visualize the particles and their tracks. During the Tanpopo mission, particles that have an orbital velocity of ~8 km/s are expected to collide with the aerogel. To simulate these collisions, we shot Deinococcus radiodurans-containing Lucentite particles into the aerogel from a two-stage light-gas gun (acceleration 4.2 km/s). The shapes of the captured particles, and their tracks and entrance holes were recorded with a microscope/camera system for further analysis. The size distribution of the captured particles was smaller than the original distribution, suggesting that the particles had fragmented. We were able to distinguish between microbial DNA and inorganic compounds after staining the aerogel with the DNA-specific fluorescence dye SYBR green I as the fluorescence of the stained DNA and the autofluorescence of the inorganic particles decay at different rates. The developed methods are suitable to determine if microbes exist at the International Space Station altitude.     

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.     

Erythrocytic nicotinamide-adenine dinucleotide phosphate levels and the genetic regulation of erythrocytic glucose 6-phosphate dehydrogenase activity in the inbred mouse

NADP measurements in erythrocytes of eight inbred strains of mice showed a correlation between low NADP and low erythrocytic G6PD levels. In vitro heat inactivation tests indicated that the NADP differences found could explain the previously demonstrated lability of G6PD in the erythrocytes of the low G6PD activity strain, C57L/J. Breeding experiments suggested a common genetic control of erythrocytic NADP levels and G6PD activity in crosses between intermediate and low strains, but an effect of sex and other variables, as yet unidentified, precluded a strong demonstration of unitary genetic control.     

Paenibacillus nicotianae sp. nov., isolated from a tobacco sample

A Gram-stain positive, facultative anaerobic endospore-forming bacterium, designated strain YIM h-19^T, was isolated from a tobacco sample. Cells were observed to be motile rods by means of peritrichous flagella and colonies were observed to be convex, yellow, circular and showed catalase-positive and oxidase-negative reactions. Strain YIM h-19^T was able to grow at 4–45 °C, pH 6.0–8.0 and 0–3 % NaCl (w/v). The predominant respiratory quinone was identified as MK-7. Major fatty acids were identified as anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The polar lipids were found to be phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unidentified polar lipids. The genomic DNA G+C content was determined to be 54 mol%. 16S rRNA gene sequence analysis showed the strain YIM h-19^T was most closely related to Paenibacillus hordei RH-N24^T and Paenibacillus hunanensis FeL05^T with similarities of 98.30 and 94.64 % respectively. However, DNA–DNA hybridization data indicated that the isolate represented a novel genomic species with the genus Paenibacillus. All data from genotypic and phenotypic analyses support the conclusion that strain YIM h-19^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nicotianae sp. nov. is proposed. The type strain is YIM h-19^T (=CGMCC1.12819^T = NRRL B-59112^T).     

Changes in cell surface anionogenic groups during differentiation ofHerpetomonas samuelpessoai mediated by dimethylsulfoxide

The surface anionic groups of untreated or dimethyl sulfoxide (DMSO)-treatedHerpetomonas samuelpessoai cells were analyzed by cell electrophoresis, ultrastructural cytochemistry, and identification of sialic acids using thin-layer chromatography. Differentiation ofH. samuelpessoai induced by DMSO treatment caused a significant increase in the net negative surface charge. In flagellates exposed to DMSO, more cationized ferritin, colloidal iron hydroxide, and sendai virus particles bound to the cell surface. Treatment of both untreated and DMSO-treated flagellates with neuraminidase decreased markedly the EPM of cells to the cathodic pole. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface ofH. samuelpessoai. Thin-layer chromatography showed thatN-acetyl andN,O-diacylneuraminic acids, in equal proportions, were present inH. samuelpessoai. However,N-acetylneuraminic acid predominates in DMSO-treated cells.     

Expression Analysis of Bombyx mori Bidensovirus Structural Proteins and Assembly of Virus-like Particles in Insect Cells

Bombyx mori bidensovirus (BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48 h post-inoculation and kept increasing until 120 h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs (vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1′, and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51 kDa (VP2) and 37 kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms.     

The properties of a few isolates of the sour cherry necrotic ringspot virus

Results of the investigation of four isolates of the sour cherry necrotic ringspot virus are presented in this paper. The isolates used caused characteristic symptoms on woody indicators “Bing”, “Montmorency”, F 12/1, and on peach seedlings. The virus was transmitted mechanically to some herbaceous species:Antirrhinum majus, Cucumis sativus, Cucurbita maxima, Chenopodium quinoa Crotalaria juncea, Momordica balsamina, Petunia hybrida andLeonorus sibiricus. The attempts to transmit the virus mechanically to further 23 herbaceous species were unsuccessful. The thermal inactivation point of the virus lies between 46 and 58°C and the dilution end-point between 10^?1 and 10^?2. The virus is stable in vitro at room temperature for more than one day. Individual virus isolates gave a positive immunological reaction with the Fulton’s “G” antiserum.     

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.)

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13^T, was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13^T belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847^T (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847^T, Streptosporangium jomthongense NBRC 110047^T (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849^T (97.6 % 16S rRNA gene similarity). A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13^T can be distinguished from S. fragile DSM 43847^T and S. jomthongense NBRC 110047^T. Moreover, strain NEAU-TWSJ13^T can also be differentiated from S. violaceochromogenes DSM 43849^T and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13^T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13^T ( = CGMCC 4.7146^T = DSM = 46724^T).     

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.