In Vitro Cell. Dev. Biol.— 36:271–276, April 2000 ᭧ 2000 Society for In Vitro Biology 1071-2690/00 $05.00+0.00

IN VITRO HOST RANGE STUDIES WITH A NEW BACULOVIRUS ISOLATE FROM THE DIAMONDBACK PLUTELLA XYLOSTELLA (L.) (PLUTELLIDAE: )

C. W. KARIUKI, A. H. MCINTOSH,1 AND C. L. GOODMAN

Department of Entomology, University of Missouri, Columbia, Missouri 65211 (C. W. K.), USDA, Agricultural Research Service, Columbia, Missouri 65203 (A. H. M., C. L. G.)

(Received 9 September 1999; accepted 12 November 1999)

SUMMARY The in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell lines. Two baculoviruses with wide host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolyhedrovirus, AcMNPV) and the celery looper (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AM1 and TN-CL1 cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV. Key words: lepidopteran cell lines; PxMNPV.

INTRODUCTION larvae. In the present report, we examined a number of lepidopteran cell lines, including a diamondback moth (DBM) line, established Over the past 10 yr the usage of lepidopteran cell lines has in- by one of us (C. W. K), for their ability to support replication of a creased considerably as a result of the development of baculovirus new baculovirus isolate recovered from infected DBM (Kariuki and expression vectors (BEVs) that are directly applicable to the bio- McIntosh, 1999). We also compared the activity and specificity of medical and agricultural fields. An important application in the field the new isolate with those of two other baculoviruses, namely of agriculture is the use of lepidopteran cell lines for the production AcMNPV (Vail et al., 1973) and AfMNPV (Hostetter and Putler, of the biopesticidal nucleopolyhedroviruses (NPVs). These baculo- 1991). viruses have been traditionally produced on a large scale in sus- ceptible larvae, but cell lines offer an attractive alternative MATERIALS AND METHODS as many studies have shown the feasibility of large-scale cell cul- ture production (Weiss and Vaughn, 1986; Maiorella et al., 1988; Insect cell lines. A cell line was established from eggs of DBM and des- Belisle et al., 1992; Agathos, 1994). There are several advantages ignated BCIRL-PX-EM1. Approximately 0.5 g of eggs were surface sterilized by immersion in 70% ethanol for 5 min followed by exposure to 0.2% sodium of in vitro systems for production of baculoviruses. These include: hypochlorite for 5 min. The eggs were then rinsed in sterile ultrapure water the purity of viral preparations free from contaminating adventitious several times followed by a final rinse in TC199-MK (McIntosh et al., 1973) agents; the process of production itself, which is less labor inten- supplemented with 10% heat-inactivated (56Њ/30 min) fetal bovine serum sive; and the selection of cell cultures which can be readily stored (FBS, Intergen Co., Purchase, NY), and containing penicillin (50 units/ml) in liquid nitrogen for future usage. Furthermore, the in vitro system and streptomycin (50 ␮g/ml). Eggs were suspended in a small volume of TC199-MK and disrupted by gentle homogenization, the suspension was cen- is more readily controlled and reproducible. The main disadvantag- trifuged at 1500 ϫ g for 10 min and the pellet was resuspended in 2.5 ml es of the in vitro system reside in the cost of production and the of growth medium and seeded into a T 12.5-cm2 flask and incubated at 28Њ generation of few-plaque mutants due to continued passage of the C. The culture medium was replaced each week by removing half and adding baculovirus resulting in decreased activity (Brown and Faulkner, an equal volume of fresh medium. The first subculture was made 3 mo after initiation of the culture and the cell line was not used in these studies until 1975; Hink and Strauss, 1976; Fraser and Hink, 1982). If the num- it had been passaged 20 times. For virus studies the growth medium was ber of passages is rigidly controlled, in vitro–produced baculovi- switched to Ex-Cell௢ 400 (JRH Biosciences, Lenexa, KS) supplemented with ruses are comparable in virulence to those produced in whole in- 10% FBS (inactivated at 56Њ C/30 min), penicillin (50 units/ml), and strep- sects (Ignoffo and Hostetter, 1974; Granados et al., 1981; Weiss et tomycin (50 ␮g/ml). Other cell lines employed in these studies are listed in al., 1992). Table 1 and include: BCIRL-HV-AM1 (Heliothis virescens), BCIRL-HS-AM1 (Heliothis subflexa), BCIRL-HZ-FB33 (Helicoverpa zea), IPLB-SF21 (Spodop- An important aspect of in vitro–produced viral insecticides is the tera frugiperda), and a clonal cell line (TN-CL1) from Trichoplusia ni (Mc- proper selection of a cell line that will produce a high concentration Intosh and Ignoffo, 1989; McIntosh, 1991). All cell lines were grown in Ex- of occlusion bodies (OBs) equivalent in activity to that produced in Cell௢ 400 with the exception of TN-CL1 which was grown in TC-199MK. Both media were supplemented with FBS and antibiotics as described above. Cell line identity. All cell lines were identified by an isoenzyme technique 1 To whom correspondence should be addressed. in conjunction with isoelectric focusing (Rice et al., 1989) using the Au-

271 272 KARIUKI ET AL.

TABLE 1

ORIGIN OF INSECT CELL LINES Species origin Tissue of Cell line of cell line origin Medium Reference

BCIRL-PX-EM1 Plutella xylostella Embryos TC199-MK Kariuki and McIntosh Ex-Cell௢400 (this report) BCIRL-HV-AM1 Heliothis virescens Pupal ovaries Ex-Cell௢400 McIntosh et al., 1981 BCIRL-HS-AM1 Heliothis subflexa Pupal ovaries Ex-Cell௢400 McIntosh and Ignoffo, 1989 BCIRL-HZ-FB33 Helicoverpa zea Fat bodies Ex-Cell௢400 Chen et al. (unpublished) IPLB-SF21 Spodoptera frugiperda Pupal ovaries Ex-Cell௢400 McIntosh and Ignoffo, 1989 TN-CL1 Trichoplusia ni Adult ovaries TC199-MK McIntosh and Ignoffo, 1989

FIG. 1. Comparison of isoenzyme banding patterns of insect cell lines described in Table 1. Extracts of cell lines were electrophoresed on preformed agarose gels for 25 min at 160 V and the gel analyzed for PGI, PGM, and LD as described in Materials and Methods.

TABLE 2

MIGRATION DISTANCES AND Rf VALUES OF ISOENZYMES Isoenzymes LD PGM PGI Cell lines Distance (mm) Rf Distance (mm) Rf Distance (mm) Rf

PXEM1 25a 20 1.14 8 1.33 29 26a 19 1.63 HZFB33 22 1.00 6 1.00 16 1.00 TN-CL1 6 0.27 2 0.33 26 1.63 HSAM1 23 1.05 3 0.50 27 1.69 SF21 24 1.09 11 1.83 31 25a 1.56 HVAM1 – – 10 1.67 11 0.69

a Underlined: main band and corresponding Rf value. thentikit௢ (Innovative Chemistry, Marshfield, MA) methodology as described C inoculated cultures were analyzed for percentage of cells showing infection by Lynn (1995). Samples were analyzed for phosphoglucose isomerase (PGI), as ascertained by the presence of OBs (McIntosh and Ignoffo, 1981), for virus phosphoglucomutase (PGM) and lactate dehydrogenase (LD). Cell line iden- titer by TCID50/ml and for the number of OBs produced (McIntosh and Ig- tity was also confirmed by a DNA amplification fingerprinting-polymerase noffo, 1989). All viral titrations (TCID50/ml) were performed in the TN-CL1 chain reaction (DAF-PCR) procedure previously described (McIntosh et al., cell line. 1996). Baculoviruses. The NPVs employed in this study included the alfalfa looper baculovirus AcMNPV (Vail et al., 1973), the celery looper virus AfMNPV RESULTS AND DISCUSSION (Hostetter and Puttler, 1991), and a new baculovirus isolate PxMNPVCL3 (Kariuki and McIntosh, 1999) from the DBM. Extracellular virus (ECV) The results of the isoenzyme analyses presented in Fig. 1 and stocks of the three baculoviruses were produced in the TN-CL1 cell line and Table 2 show that the cell lines employed in this study are clearly used in all experiments described in these studies. All NPVs were identified distinguishable from each other using PGI, PGM, and LD. This by restriction endonuclease (REN) (McIntosh and Ignoffo, 1986). Cell lines were propagated and inoculated at a multiplicity of infection of 1, as previ- finding was further confirmed using the DAF-PCR technique with ously described (McIntosh and Ignoffo, 1989). After 7 d of incubation at 28Њ primers for aldolase, prolactin, and operon-13 (Fig. 2). IN VITRO EFFECTIVITY OF A PLUTELLA XYLOSTELLA BACULOVIRUS 273

FIG. 2. DAF profiles of insect cell lines described in Table 1. Cellular DNA samples were amplified in a DNA thermal cycler using aldolase, prolactin, and operon primers in a reaction mixer. Water was run in place of DNA as a control.

REN patterns of PxMNPVCL3, AcMNPV, and AfMNPVCL1 fol- identified employing well-established techniques (Rice et al., 1989; lowing digestion of the DNAs with Hind III, XhoI, and PstI are il- McIntosh et al., 1996). In addition, the viral preparations employed lustrated in Fig. 3, and differences in the banding profiles are indi- in these studies were derived from viral isolates which were plaque- cated by arrows. The genetic similarity between these baculoviruses purified three times employing appropriate cell lines (McIntosh et is the subject of another report (Kariuki and McIntosh, 1999). al., 1985; McIntosh, 1991) before being used. This is important in The percentage of cells infected with the three baculoviruses var- reducing the possibility of viral contaminants in the inocula. ied amongst the six cell lines with the lowest percentage of 1.5% The HV-AM1 and TN-CL1 cell lines not only supported the high- recorded for PxMNPVCL3 in HZ-FB33 (Table 3) to the highest of est percentage of cells infected by the three baculoviruses, but also 99% recorded for AfMNPVCL1 in HV-AM1 (Table 5). The second gave the highest production of OBs. These findings for AcMNPV highest observed percentage of infected cells was 98% for Px- and AfMNPV support previous findings (McIntosh and Ignoffo, MNPVCL3 in TN-CL1. In general, HV-AM1 and TN-CL1 showed 1989; McIntosh, 1991) whereas the finding for PxMNPV represents the highest levels of infection of over 95% when infected with the first report for this recently discovered virus (Kariuki and Mc- AcMNPV (Table 4) and PxMNPVCL3. On the other hand, only 57% Intosh, 1999). The PX-EM1 cell line was developed with the ex- of TN-CL1 cells were infected with AfMNPVCL1. HZ-FB33 was pectation that it would provide an optimum cell culture system for completely refractile to AcMNPV and AfMNPVCL1. OB production since it would be derived from its homologous host. TN-CL1 cells produced the highest concentration of OBs (50 ϫ However, it produced the lowest number of OBs following inocu- 106/ml) followed by HV-AM1 (15 ϫ 106/ml) when infected with lation with PxMNPVCL3. Low virus production by PX-EM1 may be PxMNPV. The PX-EM1 cell line produced the lowest number of a reflection of the cell type. The cell line used in this study was OBs (0.28 ϫ 106/ml). Of the cell lines inoculated with AcMNPV, initiated from embryos and cell lines from various tissues from the HV-AM1 produced the highest number of OBs (40 ϫ 106/ml) fol- same species would have to be tested in order to determine if there lowed by HS-AM1 (17 ϫ 106/ml). Both TN-CL1 (52 ϫ 106/ml) and were any differences in virus production and thus a possible re- HV-AM1 (56 ϫ 106/ml) produced approximately equivalent num- flection of the cell type involvement. It is also known that homol- bers of OBs when inoculated with AfMNPVCL1. ogous cell lines are not always the best producers of the baculovirus

The highest ECV titer, expressed as log10 TCID50/ml, for PxMNPV isolated from the corresponding host. For example, the S. frugiperda was 7.3 as observed in HZ-FB33, HS-AM1, and HV-AM1 (Table cell line SF-21 and its clone SF-9 are extensively used by inves- 3). The highest titer for AcMNPV was 7.4 in HS-AM1 and HV- tigators in studies with AcMNPV, which was isolated from the alfalfa AM1 (Table 4). AfMNPV gave the highest titer of 8.5 in HV-AM1 looper (Autographa californica). The common practice is to name of all three baculoviruses (Table 3, 4, 5). the virus after the host from which it was isolated. However, this Baculovirus and cell line identities are important factors when may not be entirely accurate as many baculoviruses infect more examining the in vitro host range of the agent in question. In the than one species of insect, and it may just be fortuitous that the present report both the baculoviruses and the cell lines have been baculovirus in question was isolated from a particular species. In 274 KARIUKI ET AL.

FIG. 3. Agarose-gel electrophoresis of PxMNPVCL3, AcMNPV, and AfMNPVCL1 DNA digested with HindIII, XhoI, and Pst I RENs. The HindIII fragments of bacteriophage lambda DNA are included as size markers and the size (MDa) of individual bands are indicated. Arrows: →, restriction fragments unique to PxMNPVCL3, ←, fragments absent in PxMNPVCL3.

TABLE 3

REPLICATION OF PxMNPVCL3 IN LEPIDOPTERAN CELL LINES. ( ) LOWER AND UPPER 95% CONFIDENCE LIMITS. VALUES WITH THE SAME

LETTER ARE NOT SIGNIFICANTLY DIFFERENT (P Ն 0.05) BASED ON THE t-DISTRIBUTION (t2 ϭ 0.05, df ϭ n Ϫ 1 ϭ 2). ALL TITRATIONS WERE PERFORMED IN THE TN-CL1 CELL LINE

6 Cell line % Infection Log TCID50/ml Ϯ S.E. No. OB/ml ϫ 10 Ϯ S.E.

PXEM1 82.5 Ϯ 1.2 (77.3–87.7)b 5.2 Ϯ 0.1 (4.8–5.6)a 2.8 Ϯ 0.1 (2.4–3.2)d HZFB33 1.5 Ϯ 0.2 (0.67–2.3)d 7.3 Ϯ 0.0b 2.3 Ϯ 0.0e HSAM1 82.7 Ϯ 3.1 (69.4–96.0)b 7.3 Ϯ 0.0b 6.4 Ϯ 0.4 (4.7–8.1)c HVAM1 94.5 Ϯ 3.5 (79.4–109.6)a 7.2 Ϯ 0.1 (6.7–7.7)b 15.1 Ϯ 1.4 (9.1–21.1)b TN-CL1 98.1 Ϯ 1.1 (93.4–102.8)a 7.2 Ϯ 0.1 (6.8–7.6)b 49.6 Ϯ 4.3 (31.1–68.1)a SF21 57.7 Ϯ 1.8 (50.0–65.4)c 6.7 Ϯ 0.0b 12.5 Ϯ 1.2 (7.3–17.7)bc an earlier report (McIntosh and Kariuki, 1999), it was shown that of PxMNPVCL3 in low OB-producing cell lines. Both HS-AM1 and PxMNPVCL3 is related to AcMNPV and its variant AfMNPV based HV-AM1 produced the highest viral titers of ECV following inoc- on immunological and hybridization analyses. A more accurate as- ulation with AcMNPV and is supportive of a previous finding with sessment of genetic relatedness awaits sequence determination of AcMNPV in HV-AM1 (McIntosh and Ignoffo, 1989). Contrary to an genes from conserved regions such as polyh, p10, and egt. Another earlier finding, AfMNPV gave the highest titer in HV-AM1 and not poor OB-producing cell line was HZ-FB33 but it supported a high in HS-AM1, as previously reported (McIntosh, 1991). In this earlier 7.3 titer (10 TCID50/ml) of PxMNPVCL3 ECV. Thus, a high ECV titer study, the cell lines were propagated in TC-199MK and not in Ex- does not necessarily imply that the particular cell line in question Cell௢400 as was done in the present report, and it is possible that will also produce high numbers of OBs. No attempts were made to this might have affected the viral productivity. determine whether OB levels could be increased by serial passage The present investigation provides information concerning the in IN VITRO EFFECTIVITY OF A PLUTELLA XYLOSTELLA BACULOVIRUS 275

TABLE 4

REPLICATION OF AcMNPV IN LEPIDOPTERAN CELL LINES. ( ) LOWER AND UPPER 95% CONFIDENCE LIMITS. VALUES WITH THE SAME

LETTER ARE NOT SIGNIFICANTLY DIFFERENT (P Ն 0.05) BASED ON THE t-DISTRIBUTION (t2 ϭ 0.05, df ϭ n Ϫ 1 ϭ 2). ALL TITRATIONS WERE PERFORMED IN THE TN-CL1 CELL LINE

6 Cell line % Infection Ϯ S.E. Log TCID50/ml Ϯ S.E. No. OB/ml ϫ 10 Ϯ S.E.

PXEM1 60.5 Ϯ 3.5 (45.4–75.5)ab 6.2 Ϯ 0.1 (5.8–6.6)a 12.9 Ϯ 1.2 (7.7–18.1)a HZFB33 0.0 3.7 Ϯ 0.1 (3.3–4.1)b 0.0 HSAM1 78.3 Ϯ 0.9 (74.4–82.1)b 7.4 Ϯ 0.2 (6.5–8.3)a 16.7 Ϯ 0.2 (15.8–17.6)a HVAM1 94.8 Ϯ 1.1 (90.0–99.5)c 7.4 Ϯ 0.2 (6.5–8.3)a 40.4 Ϯ 6.4 (12.9–67.9)a TN-CL1 96.6 Ϯ 2.8 (84.6–108.7)c 6.9 Ϯ 0.1 (6.5–7.3)a 15.5 Ϯ 1.8 (7.5–23.5)a SF21 54.1 Ϯ 2.8 (42.1–66.1)a 6.4 Ϯ 0.1 (6.0–6.8)a 22.5 Ϯ 1.6 (3.3–48.3)a

TABLE 5

REPLICATION OF AfMNPV IN LEPIDOPTERAN CELL LINES. ( ) LOWER AND UPPER 95% CONFIDENCE LIMITS. VALUES WITH THE SAME

LETTER ARE NOT SIGNIFICANTLY DIFFERENT (P Ն 0.05) BASED ON THE t-DISTRIBUTION (t2 ϭ 0.05, df ϭ n Ϫ 1 ϭ 2). ALL TITRATIONS WERE PERFORMED IN THE TN-CL1 CELL LINE

6 Cell line % Infection Ϯ S.E. Log TCID50/ml Ϯ S.E. No. OB/ml ϫ 10 Ϯ S.E.

PXEM1 87.8 Ϯ 3.7 (71.4–103.2)abc 5.1 Ϯ 0.0 (–)a 3.7 Ϯ 0.3 (2.4–5.0)a HZFB33 0.0 4.0 Ϯ 0.1 (0.3–8.3)abc 0.0 HSAM1 86.5 Ϯ 1.0 (82.2–90.8)ac 7.7 Ϯ 0.0 (–)b 1.1 Ϯ 0.0 (–)b HVAM1 99.0 Ϯ 0.5 (96.7–101.2)b 8.5 Ϯ 0.0 (–)d 55.5 Ϯ 2.6 (44.3–66.7)c TN-CL1 57.1 Ϯ 2.0 (48.5–65.7)d 7.3 Ϯ 0.1 (6.9–7.7)bc 51.9 Ϯ 5.5 (28.2–75.6)c SF21 78.5 Ϯ 2.6 (67.3–89.7)c 7.2 Ϯ 0.0 (–)c 1.6 Ϯ 0.1 (1.16–2.0)a

vitro host range of PxMNPVCL3, as well as potential cell lines for Fraser, M. J.; Hink, W. F. The isolation and characterization of the MP and the in vitro production of this valuable pesticidal virus for the control FP plaque variants of Galleria mellonella nuclear polyhedrosis virus. Virology 117:366–378; 1982. of DBM and other lepidopteran pests (Kariuki and McIntosh, 1999). Granados, R. R.; Lawler, K. A.; Burand, J. P. Replication of Heliothis zea baculovirus in an insect cell line. Intervirology 16:71–79; 1981. Hink, W. F.; Strauss, E. Replication and passage of alfalfa looper nuclear ACKNOWLEDGMENTS polyhedrosis virus plaque variants in cloned cell cultures and larval stages of four host species. J. Invertebr. Pathol. 27:49–55; 1976. The authors express their appreciation to Drs. James Grasela and Sandra Hostetter, D. L.; Puttler, B. A new broad host spectrum nuclear polyhedrosis Brandt for helpful suggestions, Mr. Robert Pinnell, and Mr. Steve Long for virus isolated from a celery looper, Anagrapha falcifera (Kirby) (Lep- technical assistance. idoptera ). Environ. Entomol. 20:1480–1488; 1991. The senior author was supported by a scholarship from the Kenya Gov- Ignoffo, C. M.; Hostetter, D. L. Efficacy of insect viruses propagated in vivo ernment through KARI and USAID for research, which was performed in and in vitro. J. Invertebr. Pathol. 24:184–187; 1974. partial fulfillment of the requirements for the Ph.D. degree. The senior au- Kariuki, C. W.; McIntosh, A. H. Infectivity studies of a new baculovirus thor’s present address is Kenya Agricultural Research Institute, NARC Mu- isolate for the control of the diamondback moth (Plutellidae: Lepi- guga, P.O. Box 30148, Nairobi, Kenya. doptera). J. Econ. Entomol. 92(5):1093–1098; 1999. The senior author also wishes to express his gratitude to Dr. Charles Camp- Lynn, D. E. A new insect cell line from the Colorado potato beetle. In Vitro bell and staff at the University of Missouri for their administrative support. Cell. Dev. Biol. 31:91–93; 1995. Names are necessary to report factually on available data; however, the Maiorella, B.; Inlow, D.; Shauger, A.; Harano, D. Large-scale insect cell-culture USDA neither guarantees nor warrants the standard of the product, and the for recombinant protein production. Bio/Technology 6:1406–1410; 1988. use of the name by USDA implies no approval of the product to the exclusion McIntosh, A. H. In vitro infectivity of a clonal isolate of Syngrapha (Anagra- of others that may also be suitable. pha) falcifera (celery looper) multiple nuclear polyhedrosis virus. J. All programs and services of the U.S. Department of Agriculture are of- Invertebr. Pathol. 57:441–442; 1991. fered on a nondiscriminatory basis without regard to race, color, national McIntosh, A. H.; Andrews, P. A.; Ignoffo, C. M. Establishment of two con- origin, religion, sex, age, marital status, or handicap. tinuous cell lines of Heliothis virescens (F.) (Lepidoptera: Noctuidae). In Vitro 17(18):649–650; 1981. McIntosh, A. H.; Grasela, J. J.; Matteri, R. L. Identification of insect cell REFERENCES lines by DNA amplification fingerprinting (DAF). Insect Mol. Biol. 5(3):187–195; 1996. Agathos, S. N. Large scale insect cell production. In: Maramorosch, K.; Mc- McIntosh, A. H.; Ignoffo, C. M. Replication and infectivity of the single- Intosh, A., ed. Insect cell biotechnology. Boca Raton, FL: CRC Press; embedded nuclear polyhedrosis virus, baculovirus Heliothis, in ho- 1994. mologous cell lines. J. Invertebr. Pathol. 37:258–264; 1981. Belisle, B. W.; Walls, E. L.; Celeri, C.; Knoch, C.; Singer, V.; Tang, K. From McIntosh, A. H.; Ignoffo, C. M. Restriction endonuclear cleavage patterns of shake flask to industrial fermentor: challenges of large scale culture. commercial and serially passaged isolates of Heliothis baculovirus. In Vitro Cell. Dev. Biol. 28A(48A); 1992. Intervirology 25:172–176; 1986. Brown, M.; Faulkner, P. Factors affecting the yield of virus in a cloned cell McIntosh, A. H.; Ignoffo, C. M. Replication of Autographa californica nuclear line of Trichoplusia ni infected with a nuclear polyhedrosis virus. J. polyhedrosis virus in five lepidopteran cell lines. J. Invertebr. Pathol. Invertebr. Pathol. 26:251–257; 1975. 54:97–102; 1989. 276 KARIUKI ET AL.

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