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.     

Brazilian dengue virus type 1 replication in mosquito cell cultures.

The development of dengue viruses type 1 obtained from acute human sera and inoculated into mosquito cell cultures, was observed by standard transmission electron microscopy and cytochemical staining. It follows the trans-type mechanism already established of other dengue types. Direct passage of single virus particles across the cell membrane seems to be a pathway of entry and exit in dengue-1 infected cells. The nature of numerous electron translucent vesicles and tubules, produced simultaneously during virus replication inside the rough endoplasmic reticulum, was analyzed by cytochemical tests. The largest amount of virus particles was produced inside cell syncytia.     

Continuous growth of human tumor cell lines in serum-free media

Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10⁻¹⁰ M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth. This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J. Hill) and grant no. CA-37138 from the National Cancer Institute.     

S-phase-dependent enhancement of dengue virus 2 replication in mosquito cells, but not in human cells

Dengue virus (DEN) is the most prevalent cause of arthropod-borne viral illness in humans. We determined the influence of cellular growth state on DEN type 2 (DEN2) replication in mosquito and human cells, based on the hypothesis that manipulation of cellular growth state will facilitate identification of viral and cellular determinants of productive infection. Comparison of density-arrested and cycling C6/36 Aedes albopictus cells infected with a low-passage DEN2 isolate revealed that cycling cells generated higher virus titers per cell. When C6/36 cells were stalled in S-phase via a thymidine (THY) block, titers of low-passage DEN2 isolates and a high-passage strain, 16681, were increased approximately 30-fold and 10-fold, respectively. Moreover, virus release was earlier in THY-treated cells than in asynchronously cycling cells. Adsorption, entry, genome uncoating, and translation were not responsible for increased titers of virus from S-phase C6/36 cells. In contrast to the 30-fold increase in virus titers, intracellular levels of viral RNA were increased approximately 2-fold, suggesting that the S-phase-responsive step is late in the DEN2 replication cycle. Analysis of viral RNA and protein released from the cells indicated that enhanced DEN2 assembly is largely responsible for increased virus titers produced during S-phase. In contrast to C6/36 cells, DEN2 titers from S-phase human hepatoma cells or primary human fibroblasts were not increased. These results demonstrate a differential response of DEN2 to the mosquito and human cell cycle and provide a framework for detailed studies into the mechanisms mediating virus assembly.     

Cultivation of mammalian cells in serum-free media]

P3 cell lines can be grown in protein- and lipid-free synthetic medium. Using the P3 cell culture, we have shown that these cells produce autocrine growth factors and cell-substrate attachment factors. Because the cultured cells produce proteinase-inhibitor, spent medium is applicable for inactivating the action of trypsin at the time of cell passage. In addition, we have tried to cultivate various types of cells in serum-free media on the market (ASF103, ASF104 and GIT). Many cell lines can grow in these media, but inoculum dependency is observed in some cell lines. Production of monoclonal antibody by a hybridoma cell line is rather enhanced in these media. These media can be preserved at 4 degrees C or -20 degrees C for a relatively long period. These media added with EGF support the growth of Syrian hamster embryo cells at an early passage. The growth of human diploid fibroblasts in GIT medium added with EGF is a little less compared in a serum-containing medium.     

Additive protection by antioxidant and apoptosis-inhibiting effects on mosquito cells with dengue 2 virus infection

Cytopathic effects (CPEs) in mosquito cells are generally trivial compared to those that occur in mammalian cells, which usually end up undergoing apoptosis during dengue virus (DENV) infection. However, oxidative stress was detected in both types of infected cells. Despite this, the survival of mosquito cells benefits from the upregulation of genes related to antioxidant defense, such as glutathione S transferase (GST). A second defense system, i.e., consisting of antiapoptotic effects, was also shown to play a role in protecting mosquito cells against DENV infection. This system is regulated by an inhibitor of apoptosis (IAP) that is an upstream regulator of caspases-9 and -3. DENV-infected C6/36 cells with double knockdown of GST and the IAP showed a synergistic effect on activation of these two caspases, causing a higher rate of apoptosis (> 20%) than those with knockdown of each single gene (-10%). It seems that the IAP acts as a second line of defense with an additional effect on the survival of mosquito cells with DENV infection. Compared to mammalian cells, residual hydrogen peroxide in DENV-infected C6/36 cells may signal for upregulation of the IAP. This novel finding sheds light on virus/cell interactions and their coevolution that may elucidate how mosquitoes can be a vector of DENV and probably most other arboviruses in nature.     

Maintaining mosquito cell lines at high tempetatures: Effects on the replication of flaviviruses

Summary The upper thermal limit for maintenance of eleven mosquito cell lines was studied. Although most cell lines could be grown at 32°C to 34°C,Anopheles stephensi cell line could be maintained at 37°C. At higher temperatures initial growth rate was higher, but yield of cells after about a week of incubation was lower than at the standard temperature (28°C). Replication of several flaviviruses inAedes albopictus cell cultures adapted to 34.5°C was faster, and viral titers were higher than at 28°C.     

Host gene expression profiling of dengue virus infection in cell lines and patients

Background

Despite the seriousness of dengue-related disease, with an estimated 50–100 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever.

Methodology/Principal Findings

Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-κB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication.

Conclusion/Significance

Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy.     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.     

Quinic acid derivatives inhibit dengue virus replication in vitro

Background

Dengue is the most prevalent arboviral disease in tropical and sub-tropical areas of the world. The incidence of infection is estimated to be 390 million cases and 25,000 deaths per year. Despite these numbers, neither a specific treatment nor a preventive vaccine is available to protect people living in areas of high risk.

Results

With the aim of seeking a treatment that can mitigate dengue infection, we demonstrated that the quinic acid derivatives known as compound 2 and compound 10 were effective against all four dengue virus serotypes and safe for use in a human hepatoma cell line (Huh7.5). Both compounds were non-virucidal to dengue virus particles and did not interfere with early steps of the dengue virus life cycle, including binding and internalization. Experiments using a replicon system demonstrated that compounds 2 and 10 impaired dengue virus replication in Huh7.5 cells. Additionally, the anti-dengue virus effects of the quinic acid derivatives were preserved in human peripheral blood mononuclear cells.

Conclusions

Taken together, these data suggest that quinic acid derivatives represent a novel chemical class of active compounds that could be used to combat dengue virus infection.

     

Susceptibility of mosquito and lepidopteran cell lines to the mosquito iridescent virus (IIV-3) from Aedes taeniorhynchus

Mosquito iridescent viruses (MIV) are members of the genus Chloriridovirus that currently contains only the type IIV-3 from Aedestaeniorhynchus. The complete genome of invertebrate iridescent virus -3 (IIV-3) has been sequenced and the availability of a tissue culture system would facilitate functional genomic studies. This investigation, using quantitative PCR and electron microscopy, has determined that the mosquito cell lines Aedes aegypti (Aag2), Aedes albopictus (C6/36) and Anopheles gambiae (4a3A) as well as the lepidopteran cell line from Spodoptera frugiperda (SF9) are permissive to IIV-3 infection. However, IIV-3 infection remained longer in Aag2 and C6/36 cells. Virus produced in C6/36 cell line was infectious to larvae of A. taeniorhynchus by injection and per os. Ultrastructural examination of 4a3A and SF9 cells infected with IIV-3 revealed an unusual feature, where virions were localized to mitochondria. It is speculated that containment with mitochondria may play a role in the lack of persistence in these cell lines.     

Deleterious effects of hepatitis delta virus replication on host cell proliferation

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.     

Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus.

Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaviruses. In contrast, an unrelated flavivirus, yellow fever virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis virus and Semliki Forest virus are among the most distantly related alphaviruses, and our results thus indicate that mosquito cells persistently infected with Sindbis virus are broadly able to exclude other alphaviruses but that exclusion is restricted to members of the alphavirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis virus infection, by homologous virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion, which suggests a central role for the alphavirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.     

Inhibitory effects of glutathione on dengue virus production

Reduced glutathione (GSH) is the most powerful intracellular antioxidant and also involved in viral infections. The pathogenesis of dengue virus (DV) infection has not been completely clarified. This study investigated the relationship between DV serotype 2 (DV2) infections and host intracellular GSH content. Results showed infection with DV2 resulted in a decrease in intracellular GSH, which caused NF-κB activation and increased DV2 production. Supplemental GSH significantly inhibited activation of NF-κB, resulting in a decreased production of DV2 in HepG2 cells. Furthermore, high activity of NF-κB and increased production of DV2 was observed in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. In conclusion, DV2 infection could reduce host intracellular GSH concentration and benefited from this process. Supplemental GSH could inhibit viral production, indicating GSH might be valuable in the prevention and treatment of DV2 infection.