Cultured Aedes Albopictus Cells Research Articles

Dengue hemorrhagic fever in Jakarta, Indonesia in 1988: isolation of dengue virus from patient whole blood using cell cultures.

During an outbreak of dengue hemorrhagic fever (DHF) in Jakarta, Indonesia in 1988, we attempted to isolate dengue virus using mosquito cells and a medium containing heparin. Whole blood, immediately after being drawn from patients, was inoculated into Aedes albopictus cell cultures temporarily maintained in the heparin-containing medium. The overall virus isolation rate was 25% (17 of 69) samples collected within three days after admission of the patients to hospital. No virus was obtained thereafter. The successful virus isolation was apparently not related to titers of anti-dengue virus hemagglutination-inhibiting antibodies present in patients' sera. The viruses were recovered from cases of each of the four World Health Organization grades of DHF without significant differences. The technique is simple and easily performed at bedside.

Isolation of Arboviruses From Cattle and Insects at 2 Sentinel Sites in Queensland, Australia, 1979-85

Blood samples were collected regularly from two sentinel herds of cattle in northern and southern Queensland between 1979 and 1985. From 2660 samples, virus isolation attempts using baby hamster kidney (BHK21) and Aedes albopictus (AA) tissue cultures and suckling mice produced 308 viruses of which 243 (79%) were in the Palyam subgroup of orbiviruses. Mosquitoes and biting midges were collected at the southern sentinel herd site in January-February 1984 and processed for virus isolation in BHK2l and AA tissue cultures and by intrathoracic inoculation of Ae. aegypti mosquitoes. Totals of 14 338 midges of four species in 156 pools, and 9030 mosquitoes of 27 species in 232 pools, were processed and yielded 59 isolations. Of the 35 viruses isolated from Culicoides brevitarsis, 17 were members of the Palyam subgroup. Bovine ephemeral fever (BEF) virus was isolated once from Anopheles bancroftii, once from C. brevitarsis and 17 times from cattle. Akabane virus was isolated for the first time from C. wadai, as well as a further 10 times from C. brevitarsis and 20 times from cattle. Other viruses isolated from cattle included bluetongue serotype 1, and serotypes 5, 6 and 7 of epizootic hemorrhagic disease of deer (EHD). A new BEF group virus, tentatively called Oak Vale, was isolated nine times from Culex edwardsi mosquitoes. Of the orbiviruses, those in the Palyam subgroup were isolated almost exclusively in BHK2l tissue cultures but those in the bluetongue and EHD subgroups were isolated almost exclusively in AA cell cultures or after passage through Ae. aegypti. Of 22 rhabdovirus isolations from blood and insects (BEF, Kimberley and Tibrogargan), 16 were made only in AA cell cultures or after passage through Ae. aegypti.

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

The upper thermal limit for maintenance of eleven mosquito cell lines was studied. Although most cell lines could be grown at 32 degrees C to 34 degrees C, Anopheles stephensi cell line could be maintained at 37 degrees 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 degrees C). Replication of several flaviviruses in Aedes albopictus cell cultures adapted to 34.5 degrees C was faster, and viral titers were higher than at 28 degrees C.

An orbivirus of mosquitoes which induces CO2 sensitivity in mosquitoes and is lethal for rabbits.

An orbivirus, JKT-7400, isolated from Culex mosquitoes in Indonesia, replicated to a high titer and induced cytopathic effects in Aedes albopictus cell cultures. The virus produced lethal sensitivity to carbon dioxide in Culex and Aedes mosquitoes as well as in Drosophila melanogaster fruit flies but was not the agent of the hereditary sensitivity to carbon dioxide previously described for Culex quinquefasciatus. When injected intravenously in high doses, JKT-7400 virus was lethal for rabbits, apparently without replicating to a significant extent. It was not pathogenic for adult mice inoculated intravenously or for adult or suckling mice inoculated intracerebrally and intraperitoneally. Unlike an orbivirus isolated from Culex mosquitoes in China, JKT-7400 did not interfere with the replication of Japanese encephalitis virus in mosquitoes.

Ribosome metabolism during the vitellogenic cycle of the mosquito, Aedes aegypti

Ribosome accumulation and synthesis in the fat body of the mosquito Aedes aegypti increased by approx. 4-fold during 18 h after blood meal, consistent with the pattern of total RNA accumulation during the synthetic phase of the vitellogenic cycle. By 24–30 h after the blood meal, the accumulated ribosomes began to be degraded, and the total RNA content in the fat body eventually returned to previtellogenic levels. A method has been developed for isolation of ribosomal subunits from fat body, and the ribosomal proteins have been shown to have properties similar to those from cultured Aedes albopictus cells by two-dimensional polyacrylamide gel electrophoresis. Proteins S1, S10, and A from the small ribosomal subunit are phosphorylated when a fat body is incubated in Aedes saline in the presence of [ 32P]orthophosphate.

Phosphoribosylation of xanthine by extracts from insect cells

Using a sensitive TLC method, we have detected the production of xanthine monophosphate (XMP) from [ 14C]xanthine by mosquito cell extracts incubated in the presence of phosphoribosyl pyrophosphate and a phosphatase inhibitor. Extracts from both cultured Aedes albopictus cells, and from intact Aedes aegypti mosquitoes contained activity; particularly high activity was found in extracts from adult male mosquitoes. XMP-producing activity was at least 4-fold higher in extracts from cultured mosquito cells than in extracts from Drosophila melanogaster Kc cells or Spodoptera frugiperda (Lepidoptera) cells.

Marituba (Bunyaviridae) virus replication in cultured Aedes albopictus cells and in L-A9 cells.

The replication of Marituba virus (Bunyavirus genus, family Bunyaviridae), was studied in Aedes albopictus (mosquito) cells. Infection of Aedes albopictus cells with Marituba virus was characterized by an initial acute phase of infection in which large amounts of virus were produced and further by a persistent phase of infection in which virus yield was much lower. No changes in host cell DNA, RNA and protein synthesis were observed in Aedes albopictus cells infected with Marituba virus. In contrast in L-A9 (mouse fibroblasts) cells this virus shut-off the host macromolecular synthesis. During the replication of MTB virus in L-A9 cells three virus-specific proteins (G1, G2 and N) were detected. In Aedes albopictus cells, Marituba virus replicates slowly and two virus-specific proteins (G1 and N) accumulate in these cells.

Isolation of a new rhabdovirus in Australia related to Tibrogargan virus.

A virus isolated from the blood of a healthy steer and designated DPP53 was shown to have rhabdovirus morphology. Although DPP53 virus was antigenically related to Tibrogargan virus by reciprocal immunofluorescence and neutralization tests, the viruses were distinguishable by neutralization tests. DPP53 virus contained RNA and was sensitive to both ether and chloroform. The geographical distribution of neutralizing antibody to DPP53 virus in Australian cattle corresponded to the distribution of Culicoides brevitarsis indicating that this virus may be arthropod-borne with this midge as a possible vector. Antibody to DPP53 virus was detected in serum from cattle, buffalo, dogs and one horse, but not in serum from deer, pigs, humans or wallabies. Highest virus titres were obtained by growth in Vero and BHK21 cell cultures, but the virus could also be grown in Aedes albopictus cell cultures. Higher virus titres were obtained when the multiplicity of infection was low. The name advanced for DPP53 virus is 'Coastal Plains' virus.

Host-dependent mutants of Sindbis virus whose growth is restricted in cultured Aedes albopictus cells produce normal yields of virus in intact mosquitoes

Two host-dependent (hd), temperature-sensitive (ts) mutants of Sindbis virus (SV), clones 35 and 58, which are restricted in their ability to grow at 34° in cultured Aedes albopictus cells [ K. J. Kowal and V. Stollar (1981). Virology 114, 140–148] were not restricted in their ability to grow at 28–34° after intrathoracic inoculation of Ae. albopictus, Ae. dorsalis, Ae. epactius, Anopheles freeborni, and Culex tarsalis mosquitoes. Compared to standard SV (SV STD), the growth of SV clone 35 was somewhat restricted in parenterally infected C. pipiens females. Titers of SV STD and the two mutants were modulated to relative low levels by some Ae. albopictus and Ae. dorsalis females after 192 hr incubation at 34° but not at 28°, suggesting that under some conditions mosquitoes have the ability to control the replication of SV. The hd and is phenotypes of SV mutants were retained during growth at 31° for 36 hr and 72 hr in Ae. epactius and Ae. dorsalis females, respectively, and at 28° or 34° for 192 hr in Ae. albopictus females. The demonstration that mutant viral clones, which are restricted in cultured mosquito cells, can produce normal yields of virus in intact mosquitoes indicates that considerable caution must be exercised in extrapolating results obtained with cultured cells to the level of the intact organism.

Demonstration of interference between dengue virus types in cultured mosquito cells using monoclonal antibody probes.

Cultured Aedes albopictus cells (clone C6/36), persistently infected (PI) with dengue virus type 1 (dengue-1) were found resistant to superinfection with dengue virus type 3 (dengue-3). This was determined by indirect immunofluorescent (IF) staining of cultures using monoclonal antibody against a dengue-3 type-specific antigen. Dengue-1 PI cultures stained with this antibody 3 days after superinfection with dengue-3 virus (m.o.i of 2) had dengue-3 antigen in 0.1 to 1.0% of the cells. Control cultures infected with dengue-3 at the same multiplicity contained dengue-3 antigen in greater than 90% of the cells. The resistance to superinfection was not interferon-mediated, and occurred within 20 h after primary infection. In cultures simultaneously infected with two dengue virus types, one virus type was excluded from replication in most cells. A small population of cells was also found (about 1%) that contained type-specific antigen of both dengue virus types.

Sindbis virus maturation in cultured mosquito cells is sensitive to actinomycin D

In contrast to its effects on Sindbis virus production in vertebrate cell cultures, actinomycin D was found to inhibit the production of virus in cultured Aedes albopictus cells and Bombyx mori cells. One to two hours of incubation in the presence of actinomycin D was required for the inhibition to be observed. Synthesis and processing of virus structural polypeptides were found to be normal during the period of arrest of virus production. These data are interpreted as indicating the presence of a labile host function involved in the assembly of Sindbis virus components into mature virus in invertebrate host cells.

Togavirus Interference in Culex annulirostris Mosquitoes

Culex annulirostris mosquitoes infected with SFV-S, a temperature-sensitive, small-plaque mutant of Semliki Forest virus isolated from cultured Aedes albopictus cells, could not be infected later with wild-type virus (SFV-W). Interference occurred as soon as 24 h after infection with SFV-S. In mosquitoes infected with a mixture of the two viruses, both replicated normally.

Evolution and properties of Aedes albopictus cell cultures persistently infected with sindbis virus

Replicate cultures of Aedes albopictus cells were infected with Sindbis virus and then maintained for long periods of time by weekly subculture. During the first week the viral titers ranged between 10 8 and 10 9 PFU/ml, but then gradually fell and within a few weeks stabilized at about 10 5 to 10 8 PFU/ml. Such cultures were followed for the appearance of temperature-sensitive (ts) virus, small-plaque virus, and for the appearance within the cells of small (12–15 S) double-stranded viral RNA (dsRNA). Cloning experiments carried out 6 months or more after the initial infection showed that persistently infected cultures gave rise to both virus-yielding and nonyielding clones. Similar results were obtained when virus-positive clones were recloned. Prolonged treatment of persistently infected cultures with anti-Sindbis virus serum resulted in curing of the virus infection. Cured cultures behaved in every way tested as normal uninfected A. albopictus cells. The ability to cure with anti-viral serum suggests that in this system extracellular virus is needed to perpetuate the long-term infection. After persistently infected cultures were subcultured, viral RNA synthesis and viral yields were maximal during the first 3–4 days. Thereafter, although the cell number continued to increase for several days, viral RNA synthesis and viral yields both decreased sharply. This result strongly suggests that A. albopictus cells have efficient means for the regulation of viral biosynthesis. Although the resistance of persistently infected cultures to superinfection could be accounted for by interfering temperature-sensitive nondefective virus, the presence in these cells of 12 S dsRNA suggests that defective viral genomes are also present.

Failure of defective interfering particles of Sindbis virus produced in BHK or chicken cells to affect viral replication in Aedes albopictus cells.

Whereas defective interfering particles of Sindbis virus are readily produced in BHK-21 cells or chicken embryo fibroblasts by the techniques of serial undiluted passage, similar methods failed to generate such particles in Aedes albopictus cell cultures. In addition, Sindbis virus stocks produced in BHK-21 cells or chicken embryo fibroblasts and which contained defective interfering particles, when tested in A. albopictus cells, failed (i) to interfere with the replication of standard Sindbis virus and (ii) to change the pattern of intracellular viral RNA synthesis from that produced by infection with standard Sindbis virus alone. We conclude that defective interfering particles of Sindbis virus generated in chicken or hamster cells are silent or inert in mosquito cells.

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.

Observations of Aedes albopictus cell cultures persistently infected with Sindbis virus.

Annals of the New York Academy of SciencesVolume 266, Issue 1 p. 214-231 OBSERVATIONS ON AEDES ALBOPICTUS CELL CULTURES PERSISTENTLY INFECTED WITH SINDBIS VIRUS* Victor Stollar, Victor Stollar Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorThomas E. Shenk, Thomas E. Shenk Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854 Department of Biochemistry, Stanford University School of Medicine, Stanford University Medical Center, Stanford, Calif. 94305.Search for more papers by this authorRose Koo, Rose Koo Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorAkira Igarashi, Akira Igarashi Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorR. Walter Schlesinger, R. Walter Schlesinger Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this author Victor Stollar, Victor Stollar Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorThomas E. Shenk, Thomas E. Shenk Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854 Department of Biochemistry, Stanford University School of Medicine, Stanford University Medical Center, Stanford, Calif. 94305.Search for more papers by this authorRose Koo, Rose Koo Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorAkira Igarashi, Akira Igarashi Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this authorR. Walter Schlesinger, R. Walter Schlesinger Department of Microbiology College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854Search for more papers by this author First published: November 1975 https://doi.org/10.1111/j.1749-6632.1975.tb35103.xCitations: 24 * Supported by Grant GB37707 from the National Science Foundation, by Grant AI-11290 from the National Institute of Allergy and Infectious Diseases, and by the United States-Japan Cooperative Medical Science Program through United States Public Health Service Grant AI-05920. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume266, Issue1Pathobiology of Invertebrate Vectors of DiseaseNovember 1975Pages 214-231 RelatedInformation

Morphogenesis of Sindbis virus in cultured Aedes albopictus cells

Cultured mosquito cells were found to produce Sindbis virus nearly as efficiently as BHK-21 cells at 28 C. In virtually all of the cells observed in the electron microscope, virus morphogenesis was found to occur within complex vesicular structures which developed after viral infection. Viral nucleocapsids were first seen in these vesicles and appeared to be enveloped within these structures. The process of envelopment within these inclusions differed in some respects from the process previously described for the envelopment of nucleocapsids at the plasma membrane of vertebrae cells. Free nucleocapsids were only rarely seen in the cytoplasm of infected mosquito cells, and budding of virus from the cell surface was detected so infrequently that this process of virus production could not account for the amount of virus produced by the infected cells. The vast majority of extracellular virus was produced by the fusion of the virus-containing vesicles with the plasma membrane releasing mature virions and membrane nucleocapsid complexes in various stages of development.

Semliki Forest virus replication in cultured Aedes albopictus cells: studies on the establishment of persistence.

SUMMARY Semliki Forest virus (SFV) established a persistent, non-cytopathic infection in cultured Aedes albopictus cells with no effect on cumulative cell number as compared with control cultures. All cells were initially infected by SFV as judged by infective centre and immunofluorescence assay and released approx. 50 to 70 p.f.u./cell in the initial 24 h after infection. At 12 h after infection there was a ‘shut-down’ of virus-specific RNA synthesis followed by a sharp decline in the percentage of cells infected, stabilizing at about 2% by 48 h. Significant amounts of virus were released from about 2% of cells in persistently infected cultures. At 8 to 10 days a small-plaque, attenuated variant of SFV appeared in infected cultures; it replaced ‘wild-type’ virus completely by day 16. The establishment of persistence could not be related to (i) the release of interferon-like activity from infected cells, (ii) the presence of a directly antiviral agent in infected cells or medium, or (iii) changes in the total levels or subcellular distribution of acid phosphatase. Subculturing persistently infected cells stimulated cell division and promoted a (reduced) burst of virus production; simultaneous superinfection with SFV gave no increase in virus yields.

The growth of two togaviruses in cultured mosquito and vertebrate cells.

SUMMARY In cultured Aedes albopictus cells at 28 °C, Semliki Forest virus (SFV, an alphavirus; Wildy, 1971) shows a latent period, growth rate and yield per cell similar to that obtained with the same virus in three vertebrate cell lines at 37 °C. SFV and the flavivirus Kunjin both grow more slowly and to lower titre in A. aegypti cells (Peleg, 1968) than in A. albopictus cells. Kunjin grows more slowly than SFV in both mosquito and vertebrate cell lines. No replication of either virus occurred in another A. aegypti cell line (Grace, 1966). The effect of adsorption conditions on the percentage of Aedes albopictus cells infected by SFV is examined. In both mosquito and Vero cells the temperature giving the shortest latent period of SFV growth is close to the optimal temperature for cell growth. No c.p.e. were visible in infected mosquito cells; this contrasts with the invariant destruction of vertebrate cells.

Singh's Aedes albopictus cell cultures as helper cells for the adaptation of Obodhiang and kotonkan viruses of the rabies serogroup to some vertebrat cell cultures.

Multiplication of rabies serogroup viruses, Obodhiang and kotonkan, was induced in vertebrate cell cultures by using Singh's Aedes albopictus cell cultures as "helper cells."