Aedes Albopictus Mosquito Cell Line Research Articles

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60–100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20–30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study.

Mosaic composition of ribA and wspB genes flanking the virB8-D4 operon in the Wolbachia supergroup B-strain, wStr.

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes, infection interferes with replication of arboviruses, bacteria and plasmodia. Development of Wolbachia as a tool to control pest insects will be facilitated by an understanding of molecular events that underlie genetic exchange between Wolbachia strains. Here, we used nucleotide sequence, transcriptional and proteomic analyses to evaluate expression levels and establish the mosaic nature of genes flanking the T4SS virB8-D4 operon from wStr, a supergroup B-strain from a planthopper (Hemiptera) that maintains a robust, persistent infection in an Aedes albopictus mosquito cell line. Based on protein abundance, ribA, which contains promoter elements at the 5′-end of the operon, is weakly expressed. The 3′-end of the operon encodes an intact wspB, which encodes an outer membrane protein and is co-transcribed with the vir genes. WspB and vir proteins are expressed at similar, above average abundance levels. In wStr, both ribA and wspB are mosaics of conserved sequence motifs from Wolbachia supergroup A- and B-strains, and wspB is nearly identical to its homolog from wCobU4-2, an A-strain from weevils (Coleoptera). We describe conserved repeated sequence elements that map within or near pseudogene lesions and transitions between A- and B-strain motifs. These studies contribute to ongoing efforts to explore interactions between Wolbachia and its host cell in an in vitro system.Electronic supplementary materialThe online version of this article (doi:10.1007/s00203-015-1154-8) contains supplementary material, which is available to authorized users.

The insect repellent DEET (N,N-diethyl-3-methylbenzamide) increases the synthesis of glutathione S-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 [(35)S]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.

Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.

Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB

In vitro production of the obligate intracellular bacterium, Wolbachia pipientis, is essential to its manipulation as a genetic tool to spread transgenes within vector populations. We have adapted the Wolbachia-infected Aa23 Aedes albopictus mosquito cell line to Eagle's minimal medium, supplemented with nonessential amino acids, glutamine, and 20% fetal bovine serum. When plated at low densities, Aa23E cells grew as patchy monolayers, comprised of non-contiguous clusters of cells that gave rise to solid clumps of tightly adherent cells. Multicellular clumps eventually detached from the substrate and floated freely in the medium. Removal of Wolbachia by treatment with tetracycline did not alter the cytological properties of the host cells, which had a population doubling time of 4-5 d. The presence of Wolbachia was monitored by Giemsa staining of cytological preparations, polymerase chain reaction (PCR) amplification of Wolbachia 16S ribosomal DNA, and by simultaneous PCR amplification of ribosomal protein genes from Wolbachia and mosquito host cell genomes. Wolbachia morphology was pleomorphic, and Wolbachia DNA persisted in the culture medium for several weeks after degradation of PCR-amplifiable host cell DNA.

Natural aquatic insect carriers of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV)

Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). The insects were screened as potential natural carriers of MrNV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for MrNV and XSV in Belostoma sp., Aesohna sp., Cybister sp. and Notonecta sp., and negative results for Nepa sp. An Aedes albopictus mosquito cell line (C6/36) was used for infectivity assays, with viral inoculum prepared from the aquatic insects, since C6/36 cells have recently been shown to be susceptible to infection with MrNV and XSV. The C6/36 cells were harvested 4 d post-challenge for examination by electron microscopy. This revealed aggregation of viral particles throughout the cytoplasm for cells challenged with inocula from all the insect species except Nepa sp. Our results indicate that several aquatic insect species may present a risk for MrNV and XSV transmission to M. rosenbergii.

Production of mosquito densonucleosis viruses by Aedes albopictus C6/36 cells adapted to suspension culture in serum-free protein-free media.

Mosquito densonucleosis viruses (MDVs) have the potential for use as biocontrol agents. To facilitate densovirus production, the Aedes albopictus mosquito cell line C6/36 was adapted to two commercially available serum-free protein-free media (SFPFM), Sf-900 II and Drosophila-SFM. Cells adapted more slowly to growth in Sf-900 II medium, but once adapted, they grew more rapidly and appeared healthier than cells growing in Drosophila-SFM. Cells that were adapted to growth in each of these SFPFM were tested for their ability to be transfected and infected with MDVs. The Sf-900 II-adapted cell line survived transfection and showed infection rates comparable with cells growing in L15 supplemented with 10% fetal bovine serum. Cells adapted to Drosophila-SFM were less infectable and did not survive transfection. Cells adapted to each of these SFPFM were adapted to growth in spinner flasks. Cells in Sf-900 II grew substantially better in spinner flasks than cells in Drosophila-SFM media. Cells grown in Sf-900 II could be frozen and, when thawed, could support the production of densonucleosis viruses in spinner flasks.

Characterization of JNK-like protein derived from a mosquito cell line, C6/36.

When Western blot analysis of heat-killed bacteria- and lipopolysaccharide (LPS)-treated Aedes albopictus mosquito cell line C6/36 was performed using antiphospholyrated c-Jun amino-terminal kinase (JNK) antibodies, approximately 46 kDa protein was clearly detected with a peak around 30 min. After the C6/36 cells were incubated at 45 degrees C in order to induce apoptosis, the 46 kDa protein continued to be detected for at least 3 h. The internalization of fluorescein-labelled bacteria was inhibited by a JNK-specific inhibitor SP600125, suggesting that phagocytosis involves the JNK signalling pathway in mosquito cells. Based on these results, we found one candidate for the nucleotide sequence of JNK (Ae-JNK) from the C6/36 cells. This study is the first report regarding the mitogen-activated protein kinase (MAPK) of mosquito.

Detection and Identification os Parvo-like particles on a C6/36 clone of the Aedes albopictus mosquito cell line by polymerase chain reaction

Detection and Identification os Parvo-like particles on a C6/36 clone of the Aedes albopictus mosquito cell line by polymerase chain reaction

Ultrastructural differentiation of the genogroups in the genus Ehrlichia.

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.

A Parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm 3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but Jacks serological relationships with the other members of this genus.

Studies of interferon production in Aedes albopictus mosquito cells.

SummarySingh's Aedes albopictus mosquito cell line was infected with Sindbis and West Nile viruses in order to assess the ability of these invertebrate cells to produce interferon. The viruses grew...