Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Replication of Dengue Virus Type 2 in Aedes albopictus Cell Culture

The replication of type 2 dengue (D-2) virus in Aedes albopictus (Aal) mosquito cell cultures differed from that in vertebrate (LLC-MK2) rhesus monkey kidney cells. Virus readily replicated in Aal cells at either 30 or 37 C, but had no apparent effect on the host cell. Persistent infection was established with continual virus production for at least 6 months, although the virulence of progeny virus for both suckling mice and LLC-MK2 cells became attenuated. Density gradient analysis of infected Aal cell supernatant products indicated that only complete virus was released, in contrast to infected LLC-MK2 cells which also released incomplete virus. The surface antigens of the virus produced in Aal cells appeared to be considerably modified in that antiserum to vertebrate cell-produced D-2 virus did not block hemagglutination, whereas anti-Aal cell antiserum did. Virus infectivity could be neutralized by the antiserum to D-2 virus grown in vertebrate cells, however. Virus produced in LLC-MK2 cells did not demonstrate a similar host-cell modification. These results may reflect a difference in the mechanism by which D-2 virus matures in Aal cells.     

C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.     

Isolation of Rickettsia felis in the Mosquito Cell Line C6/36

We report the isolation and establishment of Rickettsia felis in the C6/36 cell line. Rickettsial growth was intense, always with 90 to 100% of cells being infected after few weeks. The rickettsial isolate was confirmed by testing infected cells by PCR and sequencing fragments of three major Rickettsia genes (gltA, ompB, and the 17-kDa protein gene).     

Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.     

Cell membrane enolase of Aedes albopictus C6/36 cells is involved in the entrance mechanism of dengue virus (DENV)

Currently, there are no antiviral drugs that effectively reduce the risks and treat the symptoms associated with dengue virus (DENV). Consequently, efforts remain primarily focused on transmission reduction. One such effort concerns DENV receptors in mosquito vectors Aedes aegypti and Aedes albopictus. Despite a lack of direct evidence demonstrating the binding of DENV to cells in mosquito vectors, one putative DENV binding protein has been α-enolase. To develop a deeper understanding, this study tested whether DENV proteins bind to enolase localized in the cytoplasmic membrane of C6/36 cells using both anti-enolase-specific antibodies, and by colocalization analysis, using confocal microscopy.Additionally, to probe the interaction of enolase with the DENV E protein, we performed a docking analysis using PatchDock and FireDock software packages. Study results demonstrate that the DENV E protein interacts with enolase in the plasma membrane of C6/36 cells of Ae. albopictus. Specific anti-enolase antibodies were found to inhibit DENV infection of these cells. Moreover, enolase was found to be localized to the cytoplasmic membrane, cytoplasm, and nucleus. These combined findings suggest that enolase participates in the entrance mechanism of DENV into vector cells.     

Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

Copper induces apoptosis in Aedes C6/36 cells

The Aedes albopictus C6/36 cell clone is used as a model system to study the effects of heavy metals on insect cells. Here we report on the effects of Cu(2+) on these cells. Similar to Cd(2+) and Hg(2+), Cu(2+) induces hyperpolymerization of the microtubules; moreover, with Cu(2+) this is followed by cell aggregation and massive apoptosis. This process, which is cell density dependent, is maximal between 0.75 and 1 mM; this is just under the LC(50) as determined by a membrane integrity test. At higher Cu(2+) concentrations, cell death occurs by necrosis. Apoptosis was ascertained by fluorescence and electron microscopy and by agarose gel electrophoresis. At 0.75 mM, apoptosis started at 18-hr exposure time and the amount of apoptotic cells increased almost linearly until 42 hr; then a plateau was reached with 70-80% apoptotic cells. This is the first report on Cu(2+)-induced apoptosis in insect cells. Possible induction mechanisms are discussed in the light of existing literature on vertebrate cells.     

The inhibitory effect of destruxin a on the replication of arboviruses in Aedes albopictus C6/36 cell line

1. Destruxin A (DxA) is a cyclic depsipeptide produced by the entomopathogenic fungus Metarhizium anisopliae. Its effect on the replication of different arboviruses in Aedes albopictus C6/36 cell line was investigated.2. DxA was effective against Chikungunya, West Nile, Ťahyňa, and Tribeč viruses. The lowest estimated virostatic concentration was 0.1 μg DxA/ml.3. The best inhibitory effect was achieved in cells pretreated with DxA before the virus application. However, an inhibitory effect was still apparent in cultures in which DxA was introduced 6 hr after virus inoculation.4. The DxA-induced virostatic effect was reversible, and the presence of the metabolite in the cultivation medium during the whole experiment was obligatory.5. The difference between the cytostatic and virostatic activities was about 500 times.     

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.     

Hemagglutinin prepared from cells of Aedes albopictus, clone C6/36, infected with type 1 dengue virus

A high titer of hemagglutinin (HAnin) was found in culture fluids of cells of Aedes albopictus, clone C6/36, infected with type 1 dengue virus. The HAnin (TC antigen) was associated with complete virions and no appreciable small-sized HAnin was produced, in contrast to the case in an infected suckling mouse brain (SMB) homogenate, in which most of the HAnin is smaller than complete virions. Extraction of the TC antigen with Tween 80-ether disrupted the HAnin into smaller-sized particles, resulting in complete loss of infectivity (TE-TC antigen), but it did not affect the titer or reactivity of the HAnin. Comparative hemagglutination-inhibition (HI) assay of human sera using TC antigen, TE-TC antigen or standard antigen extracted from infected SMB (SMB antigen) showed that TC or TE-TC antigen could be used for routine diagnostic or epidemiological HI tests instead of SMB antigen, which is rather hard to prepare.     

四种杀虫剂对白纹伊蚊(Aedes albopictus)细胞株(C6/36)的细胞遗传学效应

本文研究了四种杀虫剂:敌敌畏乳剂,灭害灵,甲基对硫磷,叶蝉散对白纹伊蚊细胞株（C6/36）染色体畸变和姐妹染色单体互换（SCE）的影响。结果表明:0.1％和0.01％浓度的敌敌畏,可使（SCE）频率明显增加,而染色体畸变率只在0.1％浓度有显著增加;0.5％的灭害灵,可使SCE频率明显增加,而染色体畸变增加不明显;甲基对硫磷和叶蝉散的实验结果表明,无论是染色体畸变和SCE频率均没有明显增加。     

Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells

Dengue virus (DENV) is naturally transmitted by mosquitoes to humans, infecting cells of both hosts. Unlike in mammalian cells, DENV usually does not cause extremely deleterious effects on cells of mosquitoes. Despite this, clustered progeny virions were found to form infection foci in a high density cell culture. It is thus interesting to know how the virus spreads among cells in tissues such as the midgut within live mosquitoes. This report demonstrates that cell-to-cell spread is one way for DENV to infect neighboring cells without depending on the “release and entry” mode. In the meantime, a membrane-bound vacuole incorporating tetraspanin C189 was formed in response to DENV infection in the C6/36 cell and was subsequently transported along with the contained virus from one cell to another. Knockdown of C189 in DENV-infected C6/36 cells is shown herein to reduce cell-to-cell transmission of the virus, which may be recovered by co-transfection with a C189-expressing vector in DENV-infected C6/36 cells. Moreover, cell-to-cell transmission usually occurred at the site where the donor cell directly contacts the recipient cell. It suggested that C189 is crucially involved in the intercellular spread of progeny viral particles between mosquito cells. This novel finding presumably accounts for the rapid and efficient infection of DENV after its initial replication within tissues of the mosquito.     

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.     

Mosquito cell line C6/36 shows resistance to Cyt1Aa6

This study aimed to investigate the resistance mechanism of C6/36 cells to Cyt1Aa6 protein under selection pressure. Receptor binding properties of Cyt1Aa6 toward sensitive and resistant C6/36 cells were investigated. More sensitive cells were detected with goat-anti-rabbit-FITC-labeled antibody, and the quantity of in vitro activated Cyt1Aa6 toxin bound to resistant cells was greatly reduced. Ligand western blot assays showed that disappearance of the 26 kDa protein and weakness of the positive bands of 68 kDa from resistant cells might lead to the resistance of C6/36 cells to Cyt1Aa6 toxin. The resistance of C6/36 cells was detected under selection in vitro-activated Cytl1Aa6 toxin. Receptor binding demonstrated that reduced Cyt1Aa6 bound to resistant cells, which might be closely related to the disappearance and weakness of some proteins. The results presented here are the first to demonstrate that Cyt1Aa protein, a uniquely characteristic toxin, induced resistance at the cellular level. It might be attributed to the change of receptors.     

Cultured mosquito cells Aedes albopictus C6/36 (Dip., Culicidae) responsive to 20-hydroxyecdysone and non-steroidal ecdysone agonist

Abstract: A cell line derived from an important mosquito, Aedes albopictus Skuse, was responsive to treatments with the insect moulting hormone 20-hydroxyecdysone (20E) and two new insecticides, RH-5849 and RH-5992, by cell clumping and inhibition of cell proliferation. The Aedes C6/36 cells showed typical numerous long extensions, and fluorescent BODIPY^®-phalloidin staining demonstrated that they resulted from F-actin polymerization. In addition, the ultrastructure of treated cells showed a high degree of vacuolarization that is in accord with protein synthesis. SDS-PAGE demonstrated that three proteins were induced that may belong to the hsp70 family. In conclusion, an insect cell line that responds to 20E may be useful for screening and identifying hormone analogues and novel insecticides.     

Single-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-Specific Transcription Factors Determine Cell Fate

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Three-dimensional structure determination of capsid of Aedes albopicus C6/36 cell densovirus

The three-dimensional structure of capsid of Aedes albopictus C6/36 densovirus was determined to 14-(A) resolution by electron cryomicroscopy and computer reconstruction. The triangulation number of the capsid is 1. There are 12 holes in each triangular face and a spike on each 5-fold vertex. The validity of the capsid and nucleic acid densities in the reconstructions was discussed.