Recovery of infective juveniles of the entomopathogenic nematode Steinernema carpocapsae via factors produced by insect cells and symbiotic bacteria

Nematology ◽  
2009 ◽  
Vol 11 (4) ◽  
pp. 611-618 ◽  
Author(s):  
Shingo Kikuta ◽  
Takashi Kiuchi ◽  
Fugaku Aoki ◽  
Masao Nagata

Abstract Entomopathogenic nematodes, Steinernema carpocapsae, show 'recovery' from the dauer form as infective juveniles (IJ) up to fourth-stage juveniles when host invasion occurs. This recovery also occurs within an insect cell line culturing system. Here we addressed the factor(s) that induce recovery. When IJ were exposed to cell medium obtained from the cultivation of Sf9 cell lines derived from armyworms (Spodoptera frugiperda), approximately 50% of IJ recovered after 4 h. By 16 h, 90% of the IJ had undergone recovery. Other insect cell lines such as silkworm (Bombyx mori)-derived BmN cells and fruit fly (Drosophila melanogaster)-derived S2 cells also secreted the recovery inducing factor(s). By contrast, mammalian cells (NIH/3T3 and HeLa) had no effect on nematode recovery. Our data also suggest that symbiotic bacteria are involved in IJ recovery; axenic IJ did not recover in the cell-cultured medium. When symbiotic bacteria isolated from IJ were propagated within the cell-cultured medium, the supernatant gained recovery-inducing activity against axenic IJ. From these results, we conclude that IJ recovery in S. carpocapsae is induced by multiple factor(s) secreted from insect cells and symbiont bacteria.

2009 ◽  
Vol 83 (18) ◽  
pp. 9113-9121 ◽  
Author(s):  
Amanda Hafer ◽  
Rebecca Whittlesey ◽  
Dennis T. Brown ◽  
Raquel Hernandez

ABSTRACT Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-β-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.


Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1702
Author(s):  
Anna Heitmann ◽  
Frederic Gusmag ◽  
Martin G. Rathjens ◽  
Maurice Maurer ◽  
Kati Frankze ◽  
...  

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.


1996 ◽  
Vol 17 (2) ◽  
pp. 165-174 ◽  
Author(s):  
F Grennan Jones ◽  
A Wolstenholme ◽  
S Fowler ◽  
S Smith ◽  
K Ziemnicka ◽  
...  

ABSTRACT Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotin-labelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r=0·96, P<0·001, n=50 and r=0·99, P<0·001, n=80 respectively). In addition, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor δ-aminolevulinic acid was included in the culture medium. Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO—TPO autoantibody complexes.


2005 ◽  
Vol 71 (8) ◽  
pp. 4833-4839 ◽  
Author(s):  
A. C. Darby ◽  
S. M. Chandler ◽  
S. C. Welburn ◽  
A. E. Douglas

ABSTRACT The cells and tissues of many aphids contain bacteria known as “secondary symbionts,” which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names “Candidatus Consessoris aphidicola” for T type and “Candidatus Adiaceo aphidicola” for U type.


1993 ◽  
Vol 10 (2) ◽  
pp. 127-142 ◽  
Author(s):  
G C Huang ◽  
M J Page ◽  
L B Nicholson ◽  
K S Collison ◽  
A M McGregor ◽  
...  

ABSTRACT Since the cloning of the TSH receptor (TSH-R), the target autoantigen of Graves' disease, the receptor has been expressed in a variety of eukaryotic cells to obtain a functional molecule. Despite this success, the levels of receptor expression have been marginally higher than the extremely low levels found in thyroid cells, preventing any progress on the purification of the molecule. In this study, the large extracellular region of the TSH-R, without the membrane spanning segments, has been expressed in insect cells using recombinant baculovirus to generate substantial quantities of the receptor protein. A monoclonal antibody previously generated to a bacterial TSH-R fusion protein was used to characterize and monitor the expression of the truncated receptor in insect cells. Two polypeptides of 63 and 49 kDa were recognized as the components of the truncated recombinant receptor. The 63 kDa protein was shown to be the glycosylated form of the smaller, 49 kDa, component. Expression in different insect cell lines showed that an increase in expression of approximately tenfold was apparent in High Five cells when compared with Sf21 cells. Very small quantities of the truncated receptor were secreted by the three insect cell lines examined, with the majority of the molecule being retained within the cells. Immunoaffinity purification of milligram quantities of the truncated receptor was achieved using the monoclonal antibody. The availability of the purified TSH-R has allowed the establishment of an enzymelinked immunosorbent assay to measure autoantibodies in the sera of patients with Graves' disease. Although the truncated receptor interacts with autoantibodies, our results show that it does not bind TSH and differs in this respect from other glycoprotein hormone receptors.


2014 ◽  
Vol 40 (6) ◽  
pp. 609-616 ◽  
Author(s):  
Tri R. Nuringtyas ◽  
Robert Verpoorte ◽  
Peter G. L. Klinkhamer ◽  
Monique M. van Oers ◽  
Kirsten A. Leiss

2014 ◽  
Vol 87 (1) ◽  
pp. 102-111 ◽  
Author(s):  
Zabihollah Shoja ◽  
Maria Tagliamonte ◽  
Somayeh Jalilvand ◽  
Yaghoub Mollaei-Kandelous ◽  
Angelo De stradis ◽  
...  

2011 ◽  
Vol 177 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Vipin Kumar Deo ◽  
Yoshitaka Tsuji ◽  
Tomomi Yasuda ◽  
Tatsuya Kato ◽  
Naonori Sakamoto ◽  
...  

2008 ◽  
Vol 133 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Jae Man Lee ◽  
Masateru Takahashi ◽  
Hiroaki Mon ◽  
Hitoshi Mitsunobu ◽  
Katsumi Koga ◽  
...  

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