Journal of Invertebrate Pathology 99 (2008) 28–34

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Journal of Invertebrate Pathology

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New cell lines derived from the black cutworm, , that support replication of the A. ipsilon multiple nucleopolyhedrovirus and several group I nucleopolyhedroviruses

Robert L. Harrison *, Dwight E. Lynn

Invasive Biocontrol and Behavior Laboratory, Plant Sciences Institute, USDA, Agricultural Research Service, Building 011A, Room 214, BARC-W, 10300 Baltimore Avenue, Beltsville, MD 20705, USA article info abstract

Article history: New cell lines were recently developed from the embryos of the black cutworm, Agrotis ipsilon (: Received 14 December 2007 ). A primary culture was initiated from 4-day-old A. ipsilon eggs in ExCell420 medium supple- Accepted 27 February 2008 mented with 5% fetal bovine serum. This initial culture produced sufficient cell growth to allow subcul- Available online 5 March 2008 tivation and eventually led to the establishment of eight distinct strains. Two of these strains (AiE1611T and AiEd6T) were selected for further characterization. Extracts of these strains were compared to an extract from A. ipsilon eggs by isozyme analysis and shown to be from the same species. Both strains were Keywords: susceptible to infection by the A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV), as well as to lepi- Agrotis ipsilon (Hufnagel) dopteran group I NPVs from A. californica, Anagrapha falcifera, Anticarsia gemmatalis, Galleria mellonella, Black cutworm Agrotis ipsilon multiple nucleopolyhedrovirus Helicoverpa armigera, Plutella xylostella, and Rachiplusia ou, with large numbers of occlusion bodies pro- AgipMNPV duced in most of the inoculated cells. The cell lines did not support the replication of group II NPVs from Baculovirus Helicoverpa zea, Lymantria dispar, and Spodoptera exigua. Both cell lines produced confluent monolayers in Nucleopolyhedrovirus plaque assays and supported the formation of plaques upon infection with AgipMNPV and Autographa Cell lines californica (Ac)MNPV. Twenty AgipMNPV plaques were picked from either AiE1611T or AiEd6T monolay- Biological control ers, and the plaque isolates were serially passaged three times through A. ipsilon cells. Only one isolate from AiE1611T cells exhibited genotypic variation in the form of an altered restriction fragment profile. Our results suggest these new lines can be useful in the study of AgipMNPV and A. ipsilon cellular and molecular biology. Published by Elsevier Inc.

1. Introduction and form the basis for environmentally benign, species-specific microbial insecticides (Hunter-Fujita et al., 1998; Moscardi, The black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: 1999). A nucleopolyhedrovirus has been identified and Noctuidae), is found in many regions worldwide and feeds on a described from the black cutworm (Boughton et al., 1999). This wide range of plants. In the United States, it is a sporadic but sig- virus, the A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV), nificant pest on corn (Clement and McCartney, 1982; Engelken has been shown in greenhouse and field trials on corn (Bough- et al., 1990; Showers et al., 1983), and is considered to be the most ton et al., 2001) and in turfgrass (Prater et al., 2006) to exhibit damaging pest of turfgrass (Potter, 1998). Management of black high insecticidal activity against A. ipsilon larvae and to reduce cutworm infestations typically involves rescue applications of larval feeding. chemical insecticides. Continuously growing insect cell lines derived from Lepidoptera Baculoviruses are a group of -specific viruses that are widely used in the study of baculoviruses and potentially can have been isolated mostly from larvae of Lepidoptera (Bonning, be developed for the generation of recombinant baculoviruses with 2005). All baculoviruses belong to a single family, the Baculovi- enhanced insecticidal activity and the large-scale production of ridae, which currently is composed of the genera Nucleopolyhe- baculovirus insecticides (Black et al., 1997; Rhodes, 1996). In this drovirus and Granulovirus (Theilmann et al., 2005). The study, two cell lines that support the replication of AgipMNPV virion-containing occlusion bodies (OBs) formed by baculovi- were developed from A. ipsilon embryos. Production and growth ruses during infection are highly virulent against host of plaque-purified AgipMNPV clones in these cells was character- ized, and the effects of passage through the cell lines was exam- * Corresponding author. Fax: +1 301 504 5104. ined. These cell lines will allow for the further study of E-mail address: [email protected] (R.L. Harrison). AgipMNPV and its development as an insecticide, and can serve

0022-2011/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.jip.2008.02.015 R.L. Harrison, D.E. Lynn / Journal of Invertebrate Pathology 99 (2008) 28–34 29 as a source of material for further studies into the physiology and 2.3. Isozyme analysis cellular and molecular biology of A. ipsilon. The AuthentikitÒ system (Innovative Chemistry, Marshfield, MA) was used for characterizing the cell lines following the manu- 2. Materials and methods facturer’s directions. Cell extracts were applied to 1% agarose gels, electrophoresed at 160 V for 25 min and then stained for malic en- 2.1. Cell line development zyme (ME, E.C. 1.1.1.40), isocitrate dehydrogenase (ICD, E.C. 1.1.1.42), phosphoglucose isomerase (PGI, E.C. 5.3.1.9), or phospho- One-day-old A. ipsilon eggs on gauze cloth were obtained from glucomutase (PGM, E.C. 2.7.5.1). The stains, buffers, and gels were the rearing colony at the Corn Insects and Crop Genetics Research obtained from Innovative Chemistry. An extract from IPLB-Sf21 Unit, Ames, IA. The eggs were incubated for 3 days at 26 °C prior cells (Vaughn et al., 1977) and another prepared from A. ipsilon to use. Primary cultures were established from 100 eggs as previ- eggs were also included on the gels for comparison. The isozyme ously described (Lynn, 1996, 2007) in Ex-Cell 420 (SAFC Biosciences, migration patterns on these gels were also compared with gels of Lenexa, KY) supplemented with 5% fetal bovine serum (FBS) and other cell lines maintained in our laboratory. 50 lg/ml gentamicin sulfate. The initial subculture was performed by replacing the culture medium with Ca/Mg-free phosphate buf- 2.4. Budded virus (BV) production fered saline that was then replaced with 50 lg/ml VMF-trypsin. After 20 min at room temperature, 2 ml fresh medium was added Black cutworm larvae were reared individually in 1-oz. cups on to the culture and the cells suspended by gentle flushing of medium A. ipsilon diet from Southland Products (Lake Village, AR) at 28 °C. from the pipet. The cell suspension was then transferred to a 12.5- When 4th-instar larvae were preparing to molt (as evident from cm2 tissue culture flask (FalconÒ) and incubated at 26 °C. After the head capsule slippage), diet was removed from the cups and the initial subcultivation, the 2.0 ml of the medium was replaced with larvae were starved overnight. The following morning, larvae that fresh medium after 1 week. Cells in the spent medium were col- had molted to 5th instar were fed an 8 mm3 cube of diet on which lected by low speed (50g) centrifugation and resuspended in fresh 8.75 Â 105 AgipMNPV polyhedra had been pipetted. Larvae that medium to create a suspended strain (designated IPLB-AiE1611S). consumed the diet cube were allowed to feed on diet ad libitum. The attached cells in the first subculture were suspended after Infected larvae at 4 days post-infection were surface-sterilized another week at 26 °C using the trypsinization procedure described with 70% ethanol and bled. Hemolymph from individual larvae in Lynn (2002) for very strongly attached cells, leading to the devel- either had a milky appearance (consistent with NPV infection) or opment of a trypsinized cell strain (IPLB-AiE1611T). contained hemocytes that carried polyhedra. Hemolymph was col- Selection favors the development of a cell line consisting of the lected from 15 larvae, pooled, and diluted 10-fold with Hink’s fast-growing cells from primary culture after relatively few pas- TNM-FH medium (SAFC Biosciences) supplemented with 10% fetal sages (Lynn, 2007). These fast-growing cells may or may not be bovine serum (Invitrogen, Carlsbad, CA) and 0.8 mg/ml glutathi- susceptible to baculovirus infection. Single-cell cloning to preserve one. Hemocytes and polyhedra were pelleted by centrifugation at cell type variety is very difficult at a low passage number. To obtain 3000g and 4 °C for 3 min, and the BV-containing supernatant was strains derived from a variety of cell types, AiE1611T cells at the stored at 0 °C. sixth passage were seeded at a low density in 3.0 ml medium in a 60-mm tissue culture dish (CorningÒ). These dishes were placed 2.5. Infections of cell lines in a plastic box with wet towels and incubated undisturbed for 4 weeks. The resulting colonies of cells were observed with an in- Two dozen cell lines being maintained in the Beltsville labora- verted phase contrast microscope and morphologically distinct tory (Table 1) and the newly established A. ipsilon cell lines were colonies marked. These colonies were scraped loose from the dish exposed to hemolymph from infected A. ipsilon larvae. Cells were with a Rainin pipet tip and transferred to 0.2 ml medium in a 96- distributed into 24-well tissue culture plates (GreinerÒ) using the well tissue culture plate (FalconÒ). Of 24 colonies initially isolated normal subculture procedure and 1.0 ml of medium supplemented in this manner, six continued growing and were maintained as un- with 50 lg/ml gentamicin for each line and 0.2 ml of a 1:10 dilu- ique strains. tion of the hemolymph was added to each cell line. The medium was replaced with 1.0 ml fresh medium after 2 h in some wells. 2.2. Cell line maintenance The cultures were incubated at 22 °C and observed periodically over a 2-week period with phase contrast and differential interfer- Regular subcultures were performed on the strains after the ence contrast microscopy for cytopathic effects second passage. AiE1611T were initially split 1:2 on a weekly basis The medium was collected from wells showing positive cyto- in 25-cm2 GreinerÒ tissue culture flasks using procedure described pathic effects and 50 ll used to inoculate the additional cells of in Lynn (2002) and Ex-Cell 420 supplemented with 5% FBS (no the same strain. These were also incubated at 22 °C and observed antibiotics were used after the cultures could be split on a regular microscopically for cytopathic effects. interval). Except when otherwise noted, Ex-cell 420 + 5% FBS is the The A. ipsilon strains selected from individual colonies were dis- medium referred to in the experiments described below with the A. tributed to 24-well plates in 0.5 ml Ex-cell + FBS inoculated with ipsilon cell lines and experiments with viruses. As the cells adapted supernatant medium from the second passage of AgipMNPV in to growth in culture the split ratios were increased, eventually AiE1611 T cells. After 2 weeks incubation at 22 °C, the medium reaching a consistent growth rate allowing a 1:15 weekly split. was suctioned from the cultures taking care to not disturb the cells The suspended strain, AiE1611S, was maintained by collecting and OBs on the bottom and the cells were disrupted by adding 0.5% medium from a mature (1- to 2-week old) culture while being (w/v) sodium dodecyl sulfate and repeated pipetting through a careful to not dislodge attached cells. The medium (containing Pasteur pipet. The plates were left on a laboratory bench overnight unattached cells) was centrifuged (50g, 10 min) and the pellet to allow OBs to settle and then counts of OBs were made under an resuspended in fresh medium and transferred to a tissue culture inverted phase contrast microscope to estimate the virus produc- flask. As with the attached strain, cells were split 1:2 during the tivity of each strain. initial subcultures but eventually could be split at 1:20 after sev- In addition to AgipMNPV, the newly established A. ipsilon cells eral months in culture. were inoculated with other nucleopolyhedroviruses. The viruses 30 R.L. Harrison, D.E. Lynn / Journal of Invertebrate Pathology 99 (2008) 28–34

Table 1 were infected with an AgipMNPV plaque diffusion in a final vol- Cell lines tested with AgipMNPV ume of 1 ml, yielding a multiplicity of infection (MOI) of approx- Species Designation Mediuma Reference imately 0.01 plaque-forming units (PFU)/cell. In addition, one Anticarsia UFL-AG286 1 Sieburth and Maruniak well each of Sf9, AiE1611T, and AiEd6T cells was infected with gemmatalis (1988) AcMNPV-C6 at a MOI of 0.5 PFU/cell. Infections were incubated Diaphania nitidalis IPLB-DmES1 1 Unpublished for 5 days at 28 °C and infected-cell medium was harvested, D. nitidalis IPLB-DmEA3 1 Unpublished clarified by centrifugation, and used for subsequent infections. Ephestia kuehniella IPLB-Ekx4S 1 Lynn and Ferkovich (2004) For the second and third passages, the cells from the two A. ipsi- 2 E. kuehniella IPLB-Ekx4T 1 Lynn and Ferkovich (2004) lon cell lines were seeded into 75- and 150-cm flasks at densi- 6 7 Helicoverpa zea BCIRL-HZ- 1 McIntosh and Ignoffo (1983) ties of 9  10 and 1.8  10 cells per flask, respectively. AM1 AiE1611T cells were infected with plaque isolates AgipMNPV- H. zea IPLB-HZ1075 1 Goodwin et al. (1982) 1T1 through À1T10 at an average MOI of 9.6 PFU/cell for the Heliothis virescens IPLB-HvE1A 2 Lynn and Shapiro (1998) second passage and 6 PFU/cell for the third passage. AiEd6T cells H. virescens IPLB-HvE1S 2 Lynn and Shapiro (1998) were infected with plaque isolates AgipMNPV-6T1 through H. virescens IPLB-HvE6A 2 Lynn and Shapiro (1998) H. virescens IPLB-HvE6S 2 Lynn and Shapiro (1998) À6T10 at an average MOI of 7.8 PFU/cell for the second passage H. virescens IPLB-HvT1 1 Lynn et al. (1988) and 4.9 PFU/cell for the third passage. As for the first passage, Lymantria dispar IPLB-LdEIta 3 Lynn et al. (1988) infected-cell media were harvested at 5 days p.i., clarified by L. dispar IPLB-LdEp 3 Lynn et al. (1988) centrifugation, and used for the next passage. AcMNPV-C6 was L. dispar IPLB-LdFB 3 Lynn et al. (1988 also passaged twice more through Sf9, AiE1611T, and AiEd6T Mamestra brassica IZD-MB0503 1 Miltenburger et al. (1977) cells. Spodoptera exigua UCR-Se1 1 Gelernter and Federici (1986) S. frugiperda IAL-SFD1 1 Lynn and Oberlander (1983) S. frugiperda IPLB-Sf9 3 Summers and Smith (1987) 2.6. Isolation and analysis of viral DNA S. frugiperda IPLB-Sf21 3 Vaughn et al. (1977) Trichoplusia ni BTI-TN5B1-4 3 Wickham et al. (1992) For AgipMNPV occluded virus (OV) DNA, A. ipsilon cadavers T. ni TN368 4 Hink (1970) resulting from AgipMNPV infection were homogenized in 0.5% T. ni IAL-TND1 1 Lynn et al. (1982) sodium dodecyl sulfate (SDS) using a Ultra-Turrax T25 mixer 2 T. ni IPLB-TN-R 4 Rochford et al. (1984) (IKA Works, Wilmington, NC). The homogenate was filtered a Medium: (1) Ex-cell 420 + 5% FBS; (2) modified TC-100 (Lynn et al., 1988); (3) through two layers of cheesecloth and a wire mesh, which were Ex-cell 420; (4) modified TNM-FH (Hink and Strauss, 1976). subsequently washed with additional volumes of 0.5% SDS. Poly- hedra were pelleted by low-speed centrifugation (750g) for 10 min. Pellets were washed by re-suspension twice in 0.1% tested are listed in Table 2. In each case, 50 ll of supernatant med- SDS and once in 0.5 M NaCl and pelleted by centrifugation after ium from the infection of permissive cell cultures was used to inoc- each washing step. Polyhedra were re-suspended in deionized ulate AiE1611T or AiE1611S cells in 1.0 ml medium in 24-well distilled H2O and solubilized with Na2CO3 as previously de- plates. The plates were incubated at 22 °C and observed micro- scribed (O’Reilly et al., 1992). scopically for cytopathic effects. The AgipMNPV OV from solubilized polyhedra and the BV from For plaque assays, 2.5 106 cells were seeded in 60 15 mm   third passage infected-cell medium were precipitated by mixing dishes for both AiE1611T and AiEd6T. Plaque assays were set up with an equal volume of 20% polyethylene glycol (PEG)–1 M NaCl using a standard method (Summers and Smith, 1987) with serial and incubating on ice overnight. The precipitated virions were pel- 10-fold dilutions of Autographa californica (Ac)MNPV-C6 and of leted by centrifugation (3000g) for 15 min. Viral DNA was the AgipMNPV BV stock prepared from larvae. Assays were incu- extracted from the virion pellets by incubation with 10 mM Tris– bated for 7 days at 28 °C. Ten AgipMNPV plaques were picked for HCl, pH 8.0–10 mM EDTA, pH 8.0–0.25% SDS–500 mg/ml protease each cell line and plaque diffusions were prepared as described K for 3 h at 37 °C. Viral DNA was purified by phenol–chloroform in Summers and Smith (1987). Plaque isolates derived from extraction and ethanol precipitation. AiE1611T cells were designated AgipMNPV-1T1 through 1T10, À The viral DNAs were analyzed by restriction endonuclease while plaque isolates derived from AiEd6T cells were designated digestion with HindIII and EcoRI. Restriction fragments were sepa- AgipMNPV-6T1 through À6T10. All plaque isolates were subjected rated by electrophoresis through a 20  25 cm 0.8% agarose gel. to two additional rounds of plaque purification. After plaque purification, AcMNPV-C6 and the AgipMNPV pla- que isolates were subjected to three cell culture passages. For 3. Results the first passage, 0.8  106 AiE1611T or AiEd6T cells were seeded into 35-mm diameter wells of 6-well plates. Individual wells 3.1. Agrotis ipsilon cell growth and morphology

The IPLB-AiE1611T line consists of tightly adherent, small, spin- Table 2 dle-shaped cells similar in morphology to mammalian fibroblasts Viruses tested on Agrotis ipsilon cell lines (described in Lynn and Ferkovich, 2004) (Fig 1A). The suspension strain, IPLB-AiE1611S is approximately Virus Abbreviation Occlusion body production the same size as the attached strain but consists of generally spher- Autographa californica MNPV AcMNPV + ical cells. Of the clonal isolates produced from AiE1611T, the strain Anagrapha falcifera MNPV AfMNPV + IPLB-AiEd6T exhibited relatively favorable growth characteristics. Anticarsia gemmatalis MNPV AgMNPV + This strain has a similar morphology to the parental AiE1611T Galleria mellonella MNPV GmMNPV + strain but has greater uniformity in cell size (Fig 1B). As is typical Heliothis armigera MNPV HearMNPV + Helicoverpa zea SNPV HzSNPV À of newly established insect cell lines, the growth rates of the A. ipsi- Lymantria dispar MNPV LdMNPV À lon cultures improved over the first year of regular subcultures, ini- Plutella xylostella MNPV PlxyMNPV + tially having approximately a doubling of cell number over a week Rachiplusia ou MNPV RoMNPV + but eventually being split at 1:15 ratio (for AiE1611T and AiEd6T) Spodoptera exigua MNPV SeMNPV À and 1:20 (for AiE1611S). R.L. Harrison, D.E. Lynn / Journal of Invertebrate Pathology 99 (2008) 28–34 31

Fig. 1. Phase contrast micrographs of A. ipsilon cell lines after 7 days growth at 26 °C. (A) 21st passage IPLB-AiE1611T cells; (B) IPLB-AiEd6T strain. Both micrographs at the same magnification; marker bar = 50 lm.

3.2. Isozyme analysis isozymes and the visibly larger quantity of malic enzyme activity in the Sf21 extract. The A. ipsilon cell line isozyme pattern was also The identical isozyme patterns observed for A. ipsilon eggs, distinguishable from those of other continuous cell lines main- AiE1611T, and AiEd6T confirm that the two cell lines were derived tained in our laboratory (Lynn, 1996; Lynn and Ferkovich, 2004). from A. ipsilon, although the egg extracts had less enzymatic activ- ity (Fig. 2). The A. ipsilon cell lines were distinguished from Sf21 3.3. Susceptibility of cell lines to AgipMNPV and A. ipsilon cells to other cells by the different mobilities of their isocitrate dehydrogenase NPVs

The initial studies screening a variety of cell lines for susceptibil- ity suggested that most of the previously available cell lines main- tained in our laboratory had no or very low susceptibility to AgipMNPV. The Sf9 and HvE6s strains showed a very low percent- age (<1%) of cells with OB-like structures (data not shown). Subse- quent inoculation of additional cultures with supernatant medium from these cultures resulted in a similarly low percentage of cells with OBs. After a couple of passages of medium in this manner, few infected cells could be found suggesting the production of prog- eny budded virus was quite low in these cultures and that these cells would not be effective tools for further study of AgipMNPV. Inoculation of the newly established cell strains yielded quite different results. AiE1611T cells inoculated with AgipMNPV re- sulted in nearly all the cells showing cytopathic effects, most with OBs (Fig 3A). Similar results were shown in the AiE1611S sus- pended strain (data not shown). Inoculation of the strains selected from colonies yielded similar results (Fig. 3B) but some strains showed greater capacity for producing OBs. In infections, AiE1611T produced 5.83 (±2.85) OBs/cell, while AiEd6T produced 8.15 (±2.50) OBs/cell. Based on superior OB production and growth characteristics, AiEd6T was selected over the other strains for fur- ther evaluation. The AiE1611T and AiE1611S strains were also susceptible to a selection of other baculoviruses. OB production was observed in 7 of the 10 viruses tested (Table 2). Only Helicoverpa zea SNPV, Lymantria dispar MNPV, and Spodoptera exigua MNPV showed no cytopathic effects on the A. ipsilon cells.

3.4. Analysis of AgipMNPV plaque isolates

Both AiE1611T and AiEd6T generated confluent monolayers in plaque assays, against which discrete plaques for AgipMNPV and Fig. 2. Isozyme analysis of new cell lines. Lane 1, extract from IPLB-AiE1611T cells; AcMNPV-C6 were easily visible under a stereomicroscope. In initial 2, IPLB-AiEd6T cells; 3, extract from Agrotis ipsilon eggs; 4, IPLB-Sf21AE cells. Gels were stained for isocitrate dehydrogenase (ICD); phosphoglucose isomerase (PGI); plaque assays of BV from infected A. ipsilon larvae, there were malic enzyme (ME); and phosphoglucomutase (PGM). approximately 7-fold less AgipMNPV plaques evident on AiEd6T 32 R.L. Harrison, D.E. Lynn / Journal of Invertebrate Pathology 99 (2008) 28–34

Fig. 3. Phase contrast micrographs of A. ipsilon cell lines inoculated with AgipMNPV. (A) IPLB-AiE1611T cells 3 days p.i. with budded virus from second passage in A. ipsilon cells. (B) IPLB-AiEd6T cells 6 days p.i. with budded virus from second passage in A. ipsilon cells. Both micrographs at the same magnification; marker bar = 50 lm. cell lawns compared to AiE1611T lawns plated with the same BV HindIII and EcoRI restriction endonucleases. Among AgipMNPV dilutions. In later infections with AgipMNPV plaque isolates, the plaque isolates plated on and passaged through AiE1611T cells, two cell lines produced similar levels of infectious virus no differences were observed among the HindIII fragment patterns (0.2À7.1 Â 107 PFU/ml from AiE1611T cells, 1.1À5.9 Â 107 PFU/ml of the AgipMNPV samples (Fig. 4A). One isolate (AgipMNPV-1T7) from AiEd6T cells). differed in its EcoRI restriction fragment pattern from the other Restriction digestion analysis was carried out to evaluate the isolates and from polyhedral AgipMNPV DNA. Compared to the integrity of AgipMNPV plaque isolate genomic DNA after cell cul- other AgipMNPV genomes, this isolate was missing a 2.6 kbp frag- ture passage and to identify possible genetic heterogeneity in virus ment and contained two extra fragments (2.3 and 2.7 kbp; Fig. 4B, from infected larvae. Viral DNA was isolated from third passage arrows) in its EcoRI fragment profile. No differences were visible AcMNPV-C6 and AgipMNPV plaque isolate BV and from AgipMNPV among HindIII and EcoR I restriction fragment patterns of OV-de- polyhedra. These DNA samples were analyzed by digestion with rived AgipMNPV DNA and DNA from the 10 isolates of AgipMNPV

Fig. 4. Restriction endonuclease digest analysis of DNA from 10 plaque isolates of AgipMNPV plated on and passaged through AiE1611T cells (AgipMNPV-1T_) or AcMNPV-C6 (AcC6) passaged through either Sf9 or AiE1611T (11T) cells. Purified DNA was digested with HindIII (A) or EcoRI (B) and electrophoresed on 0.8% agarose gels. 1-kb DNA ladder (1 kb) and k HindIII (kHIII) size standards are shown, with fragment sizes indicated on the sides of the gel. Arrows indicate novel EcoRI fragments in isolate AgipMNPV- 1T7. R.L. Harrison, D.E. Lynn / Journal of Invertebrate Pathology 99 (2008) 28–34 33 that had been plaqued and passaged through AiEd6T cells (data not single passage of the Spodoptera exigua (Se) MNPV through the S. shown). Sub-molar fragments were not observed in any of the Agi- exigua Se-UCR1 cell line resulted in the appearance of sub-molar pMNPV plaque isolate restriction digests. restriction fragments in endonuclease-cleaved DNA of progeny virus (Heldens et al., 1996). Genomic alterations in SeMNPV also were observed after five passages through other S. exigua cell lines 4. Discussion (Pijlman et al., 2003b). In contrast, DNA from all but one of the Agi- pMNPV plaque isolates yielded restriction fragment patterns that This study reports the development of A. ipsilon cell lines that were identical to that of AgipMNPV from larvae, with no sub-molar support replication of AgipMNPV, a virus for which no susceptible fragments observed. It is possible that genomic alterations are cell lines were previously available. Goodman and co-workers present in the AgipMNPV isolates, but not at a level that is readily (2001a) had previously generated two cell lines from A. ipsilon, detectable by restriction endonuclease analysis. Serial passage also AiOV (from a co-culture of ovarian tissue and fat body) and AiTS can result in alterations that are too small to be detected by restric- (from a co-culture of testes and fat body). The AiOV cell line pro- tion digest (Bischoff and Slavicek, 1997; Lua et al., 2002). 7 duced BV titers of approximately 3.5 Â 10 TCID50/ml when in- fected with Plutella xylostella MNPV, but only approximately 6% Acknowledgments of infected cells produced polyhedra (Goodman et al., 2001b). Only very low levels of BV and OV production were reported for The authors thank Jeanette Dyer (USDA-ARS, Corn Insects and AcMNPV infection of AiOV and AiTS (Goodman et al., 2001b). Ef- Crop Genetics Research Laboratory, Ames, IA) for A. ipsilon eggs forts to infect these cell lines with AgipMNPV were unsuccessful and Wendy Sparks and Dr. Bryony Bonning (Department of Ento- (Anthony Boughton, Robert Harrison, and Bryony Bonning, unpub- mology, Iowa State University) for AgipMNPV polyhedra. Mention lished observations). of trade names or commercial products in this publication is solely In addition to being susceptible to AgipMNPV infection, the for the purpose of providing specific information and does not im- AiE1611T and AiEd6T cell lines also supported replication of NPVs ply recommendation or endorsement by the US Department of from the AcMNPV and Rachiplusia ou MNPV species clusters (Jehle Agriculture. et al., 2006), both which exhibit broad host ranges in bioassays, as well as Anticarsia gemmatalis MNPV, another group I NPV. Although References AgipMNPV is a group II NPV (Jehle et al., 2006), three other group II NPVs failed to replicate in the A. ipsilon cell lines (Table 2). Bischoff, D.S., Slavicek, J.M., 1997. Phenotypic and genetic analysis of Lymantria The host range of AgipMNPV overlaps with that of the NPV and dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25K FP gene may be caused by DNA replication errors. J. Virol. 71, 1097–1106. GV isolated from the common cutworm, Agrotis segetum (AgseNPV Black, B.C., Brennan, L.A., Dierks, P.M., Gard, I.E., 1997. Commercialization of and AgseGV; Bourner and Cory, 2004; El-Salamouny et al., 2003). baculoviral insecticides. In: , L.K. (Ed.), The Baculoviruses. Plenum Press, Phylogenetic inference of selected sequences places AgipMNPV in New York, pp. 341–387. Bonning, B.C., 2005. Baculoviruses: biology, biochemistry, and molecular biology. a clade containing Polish and United Kingdom isolates of AgseNPV In: Gilbert, L., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Molecular Insect (Jakubowska et al., 2005; Jehle et al., 2006). These observations Science. Elsevier, Amsterdam, Boston, pp. 233–269. suggest the possibility that the A. ipsilon cell lines described in this Boughton, A.J., Harrison, R.L., Lewis, L.C., Bonning, B.C., 1999. 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