Viral contamination of a mosquito cell line, Aedes albopictus, associated with syncytium formation.

Viral contamination associated with syncytium formation in two sbulines of Singh's Aedes albopictus cell cultures was investigated. Electron microscopy of the syncytia revealed the presence of five different types of virus-like particles, which morphologically resembled the parvo-, picorna-, toga-, and orbi-, and bacterial viruses. When a virus-free subline of the A. albopictus cells (SL3) was inoculated with extracts of the syncytium-forming A. albopictus cells, the parvo-, toga-, and orbi-type viral agents were consistently observed. Among these three agents, the togavirus-type agent is most likely responsible for the syncytium induction. Serological examination of the infected cell extract indicated that at least one of three virus-like agents, presumably the togavirus-type agent, was related to Chikungunya. O'nyong-nyong, and Western equine encephalomyelitis viruses (alphaviruses of the Togaviridae), but separable from these.     

Cultivation of mosquito cell lines in serum-free media and their effects on dengue virus replication

Summary Seven mosquito cell lines from five species (Aedes aegypti, Ae. albopictus, Ae. pseudoscutellaris, Culex tarsalis, andToxorhynchites amboinensis) were adapted to three kinds of serum-free media (SEM), which were composed of equal volumes of tryptose phosphate broth and of either Leibovitz (L15) medium, Eagle’s minimum essential medium, or Medium 199 with Hanks’ salts. Population growth rates of the cells cultivated in the SMFs were generally slower than those of original cell cultures maintained in conventional media containing bovine sera. A karyological study showed a significant shift to heteroploidy in two of the four cell lines examined. Four SMF-adapted sublines were compared with parental cultures for replication of dengue viruses.Ae. aegypti RML-12,Ae. albopictus C6/36,Ae. pseudoscutellaris AP-61, andTx. amboinensis TRA-171 demonstrated different levels of alteration in virus replication ranging from lower titers (as inAe. albopictus C6/36) to comparable or higher titers (as inAe. aegypti RML-12) when they were simultaneously inoculated with four dengue serotypes. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.     

Replication of dengue,yellow fever,St. Louis encephalitis and vesicular stomatitis viruses in a cell line (TRA-171) derived fromToxorhynchites amboinensis

Summary The replication of seven arboviruses in a cell line (TRA-171) derived from a nonhematophagous mosquito was studied. Four serotypes of laboratory adapted and three serotypes of unadapted dengue viruses replicated in the TRA-171 cell line, inducing syncytia. The sensitivity of TRA-171 cells to dengue virus infection was comparable to that ofAedes albopictus orA. pseudoscutellaris cells. Yellow fever, St. Louis encephalitis, and vesicular stomatitis viruses also replicated. All four serotypes of dengue viruses could be plaque assayed with TRA-171 cell cultures. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.     

CulturedAedes albopictus mosquito cells synthesize hormone-inducible proteins

Summary To provide a framework for biochemical investigation of ecdysteroid action inAedes albopictus mosquito cells, we examined the effect of 20-hydroxyecdysone on cell growth and morphology, synthesis of inducible proteins (EIPs), and expression of a transfected gene regulated by a synthetic ecdysteroid response element. When cells were cultured in the continuous presence of 10⁻⁶ M 20- hydroxyecdysone, the rate of growth decreased and subtle changes in cell morphology were observed. In bothAedes aegypti andA. albopictus cells, synthesis of a small number of radiolabeled proteins, which appeared as minor bands on sodium dodecyl sulfate-polyacrylamide gels, was induced by treatment with 20-hydroxyecdysone. On two-dimensional polyacrylamide gels, 11 EIPs, ranging in size from approximately 22 to 52 kDa, were identified inA. albopictus C7-10 cells. Ten inducible proteins were localized in the cytoplasmic fraction; EIP28 and EIP31 were detected in both cytoplasmic and nuclear extracts, and EIP29 was detected only in the nucleus, at a very low level. None of these proteins corresponded to small heat shock proteins, whose genes are 20-hydroxyecdysone-inducible in someDrosophila cell lines. The juvenile hormone analog, methoprene, induced expression of a 25 kDa protein in C7-10 cells. Although 20-hydroxyecdysone sustained the synthesis of this methoprene-inducible protein, synthesis did not occur in the presence of 20-hydroxyecdysone alone. In transfectedA. albopictus cells, expression of a recombinant DNA construct containing two tandem synthetic ecdysteroid regulatory elements based on aD. melanogaster small heat shock protein gene was modestly induced by 20-hydroxyecdysone.     

Virus-like Particles Suppress Growth of the Red-Tide-Forming Marine Dinoflagellate <Emphasis Type="Underline">Gymnodinium mikimotoi</Emphasis>

We isolated 2 virus-like agents that suppressed growth of Gymnodinium mikimotoi from coastal waters of the Uwa Sea, Japan. The agents found in the flagellate cells, named GM6 and GM7, were filterable in a 0.22-lm-pore filter with approximately 100-nm shapes. Electron microscopic observation showed the presence of virus-like particles in severely damaged G. mikimotoi cells infected by GM6. The growth-suppression activity of the agents (GM6 or GM7) was lost by heating at 50°C, with treatments of DNase and protease, and filtration through a 0.05-lm filter. Our results suggest that the agents are DNA viruses infectious to and virulent for G. mikimotoi. This is the first report of a virus-like agent specific to G. mikimotoi.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

Virus infections reduce in vitro multiplication of ‘Malling Landmark’ raspberry

Summary Virus-infected plants are often symptomless and may be inadvertently used as explant sources in tissue culture research. Our objective was to determine the effect of virus infection on micropropagation. We studied the effects of single and multiple infections of three common raspberry viruses on the in vitro culture of ‘Malling Landmark’ red raspberry (Rubus idaeus L.). Virus-infected reaspberry plants were produced by leaf-graft inoculation from known-infected plants onto virus-free ‘Malling Landmark’. Single-virus source plants were infected with either tobacco streak ilarvirus (TSV), tomato ringspot nepovirus (TomRSV), or raspberry bushy dwarf idaeovirus (RBDV) and were free of other viruses as determined by enzyme-linked immunosorbent assay (ELISA) and bioassay. Virus-free, single, and multiple virus-infected ‘malling Landmark’ explants were initiated into culture and multiplied on Anderson's medium with 8.9 μM N⁶-benzyladenine (BA). At the end of the multiplication tests, ELISA reconfirmed virus infections. In vitro multiplication of ‘Malling Landmark’ was significantly reduced by multiple infections, and multiplication of plants infected with all three viruses (RBDV+TomRSV+TSV) was less than half that of virus free cultures. Shoot height and morphology of in vitro cultures were not influenced by virus infection. The greenhouse stock plant with the three-virus infection was stunted and yellow compared to the control and the other infected plants. Part of a thesis submitted by C.-W.V.T. in partial fulfilment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

Pleiotropic changes in cycloheximide-resistant insect cell clones

Summary Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. FromAedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5°C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%. This investigation was supported by grant AI20385 from the National Institutes of Health, Bethesda, MD and by a Basil O’Connor Starter Research Grant (5–415) from the March of Dimes Birth Defects Foundation.     

Molecular evidence for a novel <Emphasis Type="Italic">Coxiella</Emphasis> from <Emphasis Type="Italic">Argas monolakensis</Emphasis> (Acari: Argasidae) from Mono Lake,California, USA

Argasid ticks are vectors of viral and bacterial agents of humans and animals. Recent reports indicate that some ornithophilic argasids harbored rickettsial agents. A Nearctic tick, Argas monolakensis Schwan, Corwin, Brown is ornithophilic and has not previously been examined for rickettsial agents. Thirty adult A. monolakensis were tested by PCR for DNA from Rickettsia or Coxiella. Amplicons from a Coxiella sp. that were divergent from Coxiella burnetii were detected in 16/30 A. monolakensis. These molecular isolates were similar but not identical to C. burnetii, the Coxiella spp. of other ticks, and “Coxiella cheraxi” a pathogen of crayfish. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Evolutionary relationships of membrers of the generaTaphrina, Protomyces, Schizosaccharomyces, and related taxa within the archiascomycetes: Integrated analysis of genotypic and phenotypic characters

To study the phylogeny and evolution of archiascomycetes, we determined the full sequence of the nuclear 18S rRNA gene from 14Taphrina species and 2Protomyces species, and the partial sequence ofSchizosaccharomyces japonicus var.japonicus. The sequences were phylogenetically analyzed by the neighbor-joining, maximum parsimony, and maximum-likelihood methods. We also looked at their principal phenotypic characters and genotypic character. Relationships within the Ascomycota are concordant with the previously published phylogenies inferred from 18S rDNA sequence divergence and divide the archi-, hemi-and euascomycetes into distinct major lineages. All the trees show that, within the archiascomycete lineage, 11 of the 14Taphrina species and the 2Protomyces species are monophyletic. A core groups ofTaphrina andProtomyces is always monophyletic. The evidence from molecular and phenotypic characters such as cell wall sugar composition, ubiquinone, cell wall ultrastructure, and mode of conidium ontogeny, strongly suggests that ‘T’. californica CBS 374.39, ‘T’. maculans CBS 427.69 and ‘T’. farlowii CBS 376.39 should be excluded from the archiascomycete lineage. ‘Taphrina’ farlowii CBS 376.39 groups withCandida albicans in the Saccharomycetales, whereas ‘T’. californica CBS 374.39 and ‘T’. maculans CBS 427.69 have a basidiomycete affinity and group with Tremellalean members in the hymenomycete lineage.Schizosaccharomyces is monophyletic. The strictly anamorphic yeastSaitoella complicata groups with the apothecial ascomyceteNeolecta vitellina rather than theTaphrina/Protomyces branch.     

Indian herb ‘Sanjeevani’ (Selaginella bryopteris) can promote growth and protect against heat shock and apoptotic activities of ultra violet and oxidative stress

Selaginella bryopteris is a lithophyte with remarkable ressurection capabilities. It is full of medicinal properties, hence also known as ‘Sanjeevani’ (one that infuses life). For lack of credible scientific evidence the plant is not in active use as a medicinal herb. We provide scientific evidence for whyS. bryopteris is known as ‘Sanjeevani’. The aqueous extract ofS. bryopteris possesses growth-promoting activity as well as protective action against stress-induced cell death in a number of experimental cell systems including mammalian cells. Treatment of the cells in culture with 10% aqueous extract enhanced cell growth by about 41% in Sf9 cells and 78% in mammalian cells. Pre-treatment of cells with the Selaginella extract (SE) (1-2x5%) protected against oxidative stress (H2O2)-induced cell death. The killing potential of ultra violet (UV) was also significantly reduced when the cells were pre-treated with SE for 1 h. Thermal radiation suppressed cell growth by about 50%. Pre-treatment of cells with SE for 1 h afforded complete protection against heat-induced growth suppression. SE may possess anti-stress and antioxidant activities that could be responsible for the observed effects. Chemical analysis shows that SE contains hexoses and proteins. Taken together,S. bryopteris extract may help in stress-induced complications including those due to heat shock.     

Proteasome activity in a naïve mosquito cell line infected with <Emphasis Type="Italic">Wolbachia pipientis w</Emphasis>AlbB

We used Wolbachia pipientis strain wAlbB from Aedes albopictus Aa23 cells to infect clonal Ae. albopictus TK-6 cells, which are resistant to 5-bromodeoxyuridine. Infected TK-6 cells were cultured in medium containing 5-bromodeoxyuridine to select against Aa23 cells that might have persisted in the inoculum. Infected TK-6 lines retained the Wolbachia infection for 5 mo, indicating that their metabolic processes support Wolbachia growth and multiplication. To investigate early events after Wolbachia infection, we labeled infected cells with ³⁵S[methionine/cysteine]. Patterns of labeled proteins on sodium dodecyl sulfate gels were similar in control and infected cells, with the exception of a 29-kDa protein. Tandem mass spectrometry revealed that the 29-kDa band included α and β subunits of the 26S proteasome. Independent confirmation of the up-regulation of the proteasome was established by probing Western blots with a monoclonal antibody to the proteasome-associated co-factor, ubiquitin. Wolbachia’s loss of metabolic pathways for the synthesis of most amino acids and retention of pathways for their uptake and metabolism suggest that proteasome activation provides a mechanism whereby controlled degradation of intracellular host proteins would increase availability of amino acids to support establishment and maintenance of the Wolbachia infection.     

Hepatopancreas cell cultures from mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz’ L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2–3 d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.     

Increased levels of the cell cycle inhibitor protein, dacapo, accompany 20-hydroxyecdysone-induced G1 arrest in a mosquito cell line

When treated with the steroid hormone 20‐hydroxyecdysone (20E), C7‐10 cells from the mosquito, Aedes albopictus, arrest in the G1 phase of the cell cycle. To explore whether 20E‐mediated cell cycle arrest proceeds through increased levels of cell cycle inhibitor (CKI) proteins, we cloned the Ae. albopictus homolog of dacapo, the single member of the Cip/Kip family of CKI proteins known from Drosophila melanogaster. The Ae. albopictus dacapo cDNA encoded a 261‐amino acid homolog of the Aedes aegypti protein XP_001651102.1, which is encoded by an ～23 kb gene containing three exons. Like dacapo from D. melanogaster, the ～27 kDa protein from Aedes and Culex mosquitoes contained several S/TXXE/D motifs corresponding to potential protein kinase CK2 phosphorylation sites, and a binding site for proliferating cell nuclear antigen (PCNA). When extracts from cells treated with 20E were analyzed by western blotting, using a primary antibody to synthetic peptides from the mosquito dacapo protein, up‐regulation of an ～27 kDa protein was observed within 24 h, and the abundance of the protein further increased by 48 h after hormone treatment. This is the first investigation of a cell cycle inhibitory protein in mosquitoes. The results reinforce growing evidence that 20E affects expression of proteins that regulate cell cycle progression. © 2011 Wiley Periodicals, Inc.     

Transinfection and growth discrepancy of Drosophila Wolbachia strain wMel in cell lines of the mosquito Aedes albopictus

Aim: The Wolbachia strain wMel can protect Drosophila melanogaster against pathogenic RNA viruses. To analyse the potential of this inhibitory effect against arboviruses vectorized by these mosquitoes, we here first transinfected the Aedes albopictus Aa23 and C6/36 cell lines with the Wolbachia strain wMel and then monitored their infection dynamics. Methods and Results: Wolbachia strain wMel was transferred into A. albopictus Aa23 and C6/36 cell lines using the shell vial technique. The presence of the bacterium in the transinfected cells was monitored by quantitative PCR and fluorescence in situ hybridization. Bacteria could be detected in the cytoplasm of both the Aa23 and C6/36 cell lines. However, the dynamics and stability of the bacterial infection differed depending on the initial cell background. The Aa23 cell line, which had been treated with a tetracycline antibiotic 2 years previously to eliminate its natural Wolbachia wAlbB‐infecting strain, lost the introduced Wolbachia wMel strain after 12 passages postinfection. In contrast, the C6/36 cell line, which had originally been aposymbiotic, displayed a stable infection with Wolbachia wMel. The bacterial density in C6/36 was greater than that of the A. albopictus RML12 cell line from which the wMel strain had originated. Conclusions: Transient or persistent transinfection of A. albopictus Aa23 and C6/36 cell lines with Wolbachia wMel strain was achieved. The results indicate the influence of the genetic background of mosquito cells in maintaining Wolbachia originating from a distant dipteral host. Significance and Impact of the Study: The cell model built here can now be used to investigate the viral inhibitory effect of the Wolbachia wMel strain against arboviruses such as dengue and chikungunya, which are transmitted by the mosquito A. albopictus.     

Tomato-aphid-hoverfly: a tritrophic interaction incompatible for pest management

Trichome-based tomato resistance offers the potential to reduce pesticide use, but its compatibility with biological control remains poorly understood. We evaluated Episyrphus balteatus De Geer (Diptera, Syrphidae), an efficient aphidophagous predator, as a potential biological control agent of Myzus persicae Sulzer (Hemiptera, Aphididae) on trichome-bearing tomato cultivars. Episyrphus balteatus’ foraging and oviposition behavior, as well as larval mobility and aphid accessibility, were compared between two tomato cultivars (Lycopersicon esculentum Mill. ‘Moneymaker’ and ‘Roma’) and two other crop plants; broad bean (Vicia faba L.) and potato (Solanum tuberosum L.). Hoverfly adults landed and laid more eggs on broad beans than on three species of Solanaceae. Hoverfly larval movement was drastically reduced on tomato, and a high proportion of hoverfly larvae fell from the plant before reaching aphid prey. After quantifying trichome abundance on each of these four plants, we suggest that proprieties of the plant surface, specifically trichomes, are a key factor contributing to reduced efficacy of E. balteatus as a biological agent for aphid control on tomatoes. Handling editor: Stanislaw Gorb     

The insect repellent DEET (<Emphasis Type="Italic">N,N</Emphasis>-diethyl-3-methylbenzamide) increases the synthesis of glutathione <Emphasis Type="Italic">S</Emphasis>-transferase in cultured mosquito cells

DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET’s repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET’s toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [³⁵S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS–polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.     

Characterization of a novel antimycotic agent,cinnamyl benzoate,using yeast-phase Sporothrix schenckii

Cinnamyl benzoate specifically inhibited the growth of yeast-phase cells of the pathogenic fungus Sporothrix schenckii. A commercially available antimycotic agent, miconazole nitrate, released large amounts of K⁺ and P_i from S. schenckii probably due to it damaging the cell membrane, but no such release was observed with cinnamyl benzoate or with another commercial antimycotic agent, Tolnaftate. The sterol content of cells treated with cinnamyl benzoate and Tolnaftate was decreased and large amounts of squalene accumulated in the cells. Cinnamyl benzoate may therefore inhibit sterol synthesis in S. schenckii.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, lkeda 4-22-1, Kumamoto 860, Japan.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.