Replication of Heliothis zea nuclear polyhedrosis virus in cloned cell lines

Clones from two Heliothis zea (Lepidoptera:Noctuidae) ovarian cell lines, BCIRL-Hz-AM1 (AM1) and BCIRL-Hz-AM3 (AM3), were generated and their ability to produce H. zea nuclear polyhedrosis virus (HzSNPV) was compared. Titers of extracellular virus (ECV) ranged from 5.5 (AM3-F9) to 44.9 x 10(5) PFU/ml (AM1-A4), with the parental cell lines AM3 and AM1 producing 14.8 and 26.4 x 10(5) PFU/ml, respectively. Concentrations of polyhedral inclusion bodies (PIB) produced by the cloned lines ranged from 0.7 (AM3-F9) to 59.6 x 10(6) PIB/ml (AM1-B3), while the parental cell lines generated 6.5 (AM3) and 12.9 x 10(6) PIB/ml (AM1). The percentage of cells from the cloned lines that produced PIB ranged from 39 to 86.4% for AM3-F9 and AM1-A7, respectively, with the parental lines exhibiting 49.1% (AM3) and 75.3% (AM1) cells with PIB. The number of PIB per cell also differed markedly between cell lines, varying from 18.3 (AM3-F9) to 184.4 (AM1-B3) PIB/cell. The parental lines produced 57.3 (AM3) and 75.9 (AM1) PIB/cell. Thus, significant differences were seen in virus production (ECV, PIB) between parental cell lines, as well as between parental cell lines and their clones. In addition, cell lines were characterized with regard to their growth rates and isoenzyme patterns.     

Replication and infectivity of the single-embedded nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines

Three cell lines of Heliothis zea and one cell line of Heliothis virescens replicated the singleembedded, nuclear polyhedrosis virus (NPV) of H. Zea, (i.e., Baculovirus heliothis) with concomitant production of polyhedral inclusion bodies (PIB). Between 20 and 60% of the H. zea cells produced PIB, whereas only 3% of H. virescens cells were found to produce PIB. The H. zea cell lines produced 10 to 20 times more PIB than did the H. virescens cell line. The PIB from all cell lines produced typical symptoms of an NPV infection when bioassayed against larvae of H. zea. More than 99% of the total viral activity of the final whole culture was due to the PIB.     

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Replication ofAutographa californica baculovirus (AcMNPV) in a coleopteran cell line

Summary A coleopteran cell line (AGE) derived from the cotton boll weevilAnthonomus grandis supported replication ofAutographa californica multiple nuclear polyhedrosis virus (AcMNPV). The titer of extracellular virus (ECV) and the number of occlusion bodies (OB) produced in AGE cells were approximately equal to those produced by aTrichoplusia ni cell line (TN-CL1), and the OB produced by both cell lines were equally infectious forT. ni larvae. The identity of the AGE cell line was established by chromosome and isoenzyme analyses.     

In vitro production studies with a wild-type Helicoverpa baculovirus

The potential use of a wild-type Helicoverpa baculovirus as a biopesticide, using insect cell culture for its production, has been investigated. A Helicoverpa zea cell line was adapted to grow in suspension culture using a serum-free medium, SF900II and serum supplemented SF900II. The serum supplemented cells were infected with a wild-type nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV), at different stages of growth, in conditioned and tresh medium, to determine the effect of cell density on polyhedra production. Cultures infected at low cell densities, produced similar yields of virus (20–40 PIB/cell), irrespective of medium conditions. However, in infections which occurred at high cell densities, there was a 16-fold improvement in cell specific yields, when the spent medium was renewed with fresh medium prior to infection. Results indicated that only 60–70% of the viable cells in a culture produced polyhedra as a result of infections.     

Establishment of a lepidopteran hybrid cell line by use of a biochemical blocking method

Summary We report here on the establishment of aCydia pomonella (Cp) hybrid cell line IZD-Cp 4/13. As there have been no reports on somatic cell fusion involving lepidopteran cell lines so far, we had to develop an appropriate fusion procedure. We first tried—but without much success—to obtain HAT (hypoxanthine, aminopterin and thymidine)- or TAM (thymidine, adenine, and methotrexate)-selectable strains of the threeCydia pomonella cell lines IZD-Cp 2202, IZD-Cp 0508 and Cp 169. We then tried and succeeded in developing a fusion procedure based on the use of biochemically blocked permanent cells as one partner in the fusion. Biochemically inhibited IZD-Cp 2202 cells and embryonic Cp cells were hybridized by polyethyleneglycol treatment. The cells of the hybrid cell line IZD-Cp 4/13 differ from the permanent parental cells (cell line IZD-Cp 2202) with respect to morphology, DNA content isoenzyme patterns, and response to challenge with theChoristoneura murinana nuclear polyhedrosis virus. This work was supported by the Bundesministerium fuer Forschung und Technologie, Bonn, FRG.     

Characterization of clonal populations of theHeliothis zea cell line IPLB-HZ 1075

Summary A total of 24 clones (HZ 1075/UND-A through X) were initially isolated by dilution plating from the established IPLB-HZ 1075 cell line. Many of the isolates with highly vacuolated cytoplasms eventually died during subculturing. The cloned cell strains differed in their predominant morphology, cell doubling times, and relative ability to support replication of the singly encapsulated nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). The origin of the cloned cell strains was confirmed by comparing their isozyme profiles with those of the parental IPLB-HZ 1075 cell line andH. zea larvae using stains for fumerate hydratase, lactate dehydrogenase (LDH), and malic dehydrogenase (MDH). One dipteran and several lepidopteran cell lines maintained in our lab were also separable using stains for LDH and MDH. This research was supported in part by USDA grant number GAM 8400211, and by a Jesse Jones Faculty Research Award from the University of Notre Dame to M. J. F.     

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth (growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five™ cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium (LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

Summary A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.     

Microbial communities of Solanum tuberosum and magainin-producing transgenic lines

Antimicrobial peptide magainin II, isolated from the skin of the African clawed toad, has shown activity in vitro against a range of micro-organisms. Transgenic potato lines expressing a synthetic magainin gene show improved resistance to the bacterial plant pathogen, Erwinia carotovora. Culturable bacterial and fungal communities associated with magainin-producing potato plants were compared with those communities from the non-transgenic parental control and with another potato cultivar. Total numbers of aerobic bacteria recovered from the leaves of the magainin-producing line, its non-transgenic parent line and an unrelated cultivar did not differ significantly. There were no detectable differences in the numbers of Gram-positive and Gram-negative bacteria, pseudomonad populations or fungi recovered from foliage from the three plant lines. Bacterial populations recovered from the roots of a magainin-expressing plant line did not differ significantly from populations recovered from the unmodified parental line. Tubers from the magainin-expressing transgenic potatoes, however, had significantly lower total numbers of bacteria than tubers produced by unmodified plants. In vitro testing of rhizosphere isolates against magainin analogues found that bacterial isolates varied in their susceptibility to the peptides. There were no significant differences in the total numbers of fungi and yeasts recovered from the various plant lines, with one exception: higher numbers of fungi were recovered from roots of magainin-expressing plants than the unmodified control plants.     

Comparison of in vivo infectivity of tissue-cultured nonoccluded virus and alkali-liberated occluded virus of Baculovirus heliothis

Feeding and intrahemocelic injection studies using tissue-culture-derived-nonoccluded virus (TCNOV) and occluded virus liberated by alkaline solution (ALOV) from polyhedral inclusion bodies were conducted with the single-embedded Heliothis nuclear polyhedrosis virus, Baculo-virus heliothis (H_zSEV). Comparisons of infectivity between ALOV and NOV were based upon the number of adminstered plaque-forming-units (PFU). There was little, if any, difference in infectivity between ALOV and TCNOV of H_zSEV when injected into 4th-instar larvae of Heliothis virescens. The LD₅₀, from the multiple dose injection studies, for ALOV and TCNOV was 6.5 ± 1.2 PFU per larva and 3.4 ± 0.9 PFU per larva, respectively. Injection of a single dose (5 PFU per larva) resulted in a larval mortality of 83.2 ± 3.4 and 62.6 ± 5.7% for ALOV and TCNOV of the H_zSEV, respectively. The LC₅₀ of ALOV and TCNOV, from the multiple-dose feeding tests, was 3.1 ± 0.4 PFU/cm² and 4.5 ± 0.9 PFU/cm², respectively. Feeding 24-hr-old larvae on virus-treated diets at a single dose (50.0 PFU/cm²) resulted in a 1.5-fold difference in percentage larval mortality between ALOV (91.0 ± 4.0%) and TCNOV (61.2 ± 3.0%). Counts of viral particles (VP), based upon electron microscopy, were 14.3 ± 2.6 × 10¹⁰ and 5.2 ± 1.1 × 10⁷ VP/ml for the ALOV and TCNOV, respectively. Thus, each larva ingesting or injected with one PFU received ca. 3500 × more VP of ALOV than in did of TCNOV.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

Reciprocal transfer of male sterile and normal plasmons in Petunia

Summary The goal in this experiment was to achieve direct plasmon transfer via cell fusion. Two lines were used — a normal fertile line of P. hybrida, and a cytoplasmic male sterile (cms) line with the nuclear background of P. parodii. Two plants phenotypically similar to the original male sterile line were developed from protoplasts, but instead of being cms they were male fertile. On the other hand, two plants typical of the original normal line developed from protoplasts, but they were cms instead of fertile. Chromosome counts were done and in all cases the expected diploid number (=14) was found. Genetic analysis showed that sorting out of cms and fertile segregants was evident in the first and second backcross of the cms cybrids. The fertile type cybrids were stable fertile for several generations of selfing and proper backcrossing. These results are discussed in the light of an earlier fusion experiment in which these two parental lines were involved.Contribution from the Department of Plant Genetics and Breeding, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 991-E, 1984 series     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

Establishment,growth kinetics,and susceptibility to AcMNPV of heat tolerant lepidopteran cell lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1–4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

Asymmetric protoplast fusion aimed at intraspecific transfer of cytoplasmic male sterility (CMS) in Lolium perenne L.

Summary Techniques have been developed for the production of cybrids in Lolium perenne (perennial ryegrass). Gamma-irradiated protoplasts of a cytoplasmically male-sterile breeding line of perennial ryegrass (B200) were fused with iodoacetamide-treated protoplasts of a fertile breeding line (Jon 401). After fusion 25 putative cybrid calli were characterized to determine mitochondrion type and composition of the nuclear genome. Analysis of phosphoglucoisomerase isozyme profiles and determination of the ploidy level by flow cytometry indicated that all of the calli tested essentially contained the nuclear DNA of the fertile line. However, the presence of parts of the nuclear DNA from the sterile line could not be excluded. Southern blotting of total DNA isolated from the parental lines and putative cybrids combined with hybridizations using the mitochondrial probes cox1 and atp6 revealed that the mitochondria of the calli originated from the fertile line (5 calli), the sterile line (5 calli) or from both parental lines (15 calli). The hybridization patterns of the mtDNA from the cybrid calli showed extensive quantitative and qualitative variation, suggesting that fusion-induced inter- or intramolecular mitochondrial recombination had taken place.     

The role of adenosine 3′,5′-monophosphate in the transformation of cloudman mouse melanoma cells

The role of adenosine 3′,5′-monophosphate (cyclic AMP) in the regulation of mouse melanoma cell growth and differentiation was investigated. A variant melanoma (Cloudman S91-F) which displays a greater degree of transformation than the parental cell (Cloudman S91) was isolated. A correlation between cyclic AMP metabolism and transformation was made. Dibutyryl cyclic AMP depressed cell growth and increased pigmentation in both parental and variant cell lines. The parental cell line, however, was more responsive to melanocyte-stimulating hormone (MSH) which was found to affect cell growth and pigmentation by increasing cyclic AMP levels. The more transformed S91-F cell line contained lower levels of cyclic AMP than the parental cell line, and this fact correlated well with the higher degree of growth and lesser degree of pigmentation in the variant. Enzymatic analysis revealed that the hydrolysis of cyclic AMP in both cell lines was similar, while the adenylate cyclase activity of the variant cell line was lower than that of the parental cell line. Lineweaver-Burk plots demonstrated that the K_m′s for the enzymes in the two cell lines were the same but that the V_max of the S91-F cell line was significantly less than that of the S91 cell line. Thus, the lesion in the S91-F cell which is responsible for its more transformed characteristics seems to be one which affects adenylate cyclase at the level of the cell membrane.