Continuous cell line from Spodoptera exigua (Lepidoptera: Noctuidae) that supports replication of nuclear polyhedrosis viruses from Spodoptera exigua and Autographa californica

A continuous cell line, designated UCR-SE-1, has been established from larvae of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). The cell line was established from minced neonate larvae treated with collagenase, and is grown in a modified TNM-FH medium with an osmotic pressure of 400 mOsm. The cell line consists of a mixture of two cell types, epithelial-like cells and spindle-shaped cells, both of which grow as attached monolayers. The cell line has a population doubling time of 56 hr, and has undergone more than 100 serial passages. Greater than 90% of the spindle-shaped cells support replication of the multiple nucleocapsid nuclear polyhedrosis viruses from Spodoptera exigua and Autographa californica, although the epithelial-like cells support replication of the latter virus only.     

Effects of Temperature and pH on Survival of Free Nuclear Polyhedrosis Virus of Autographa californica

The effects of temperature and low pH on replication and survival of nonoccluded Autographa californica nuclear polyhedrosis virus were investigated. No virus replication or formation of polynuclear inclusion bodies occurred at 37°C. The virus was immediately inactivated upon exposure to pH 2.0 and was inactivated within 1 h at pH 4.0. The virus titer slowly declined, a 3-orders of magnitude reduction in virus titer, at pH 5.0 during a 4-h exposure. Virus survival at pH 6.0 was equal to that of the control in cell culture medium 199 MK (pH 7.12).     

The influence of the condition of cells and medium on production of polyhedra of Autographa californica nuclear polyhedrosis virus in vitro

A culture of an established cell line of Trichoplusia ni (TN-368) was infected with the nuclear polyhedrosis virus of Autographa californica (ACNPV) at different stages of growth. After 50 hr of growth (early log phase), production of polyhedral inclusion bodies (PIBs) declined rapidly as cell number increased. Medium taken from cultures in the later stages of growth suppressed PIB production in early log-phase cells. Cultures infected with ACNPV and inoculated into fresh medium at high cell concentrations produced relatively fewer PIBs than those inoculated at lower concentrations. The depressive effect of utilized medium is suggested to be the result of exhaustion of a precursor rather than accumulation of an inhibitor.     

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.     

Plaque Assay of Nuclear Polyhedrosis Viruses in Cell Culture

The nuclear polyhedrosis virus of Autographa californica has been titrated in Spodoptera frugiperda cells by the plaque method, using a solid overlay which does not require either the use of modified culture medium or expensive purified agarose or the addition of culture medium as a liquid layer above the solid agarose. This assay is more sensitive than that using a viscous methyl cellulose overlay but less sensitive than the end-point dilution technique. Neither Trichoplusia ni nor Bombyx mori cells were satisfactory as indicators for the assay as described, since they failed to form a stable monolayer. Manduca sexta cells could be utilized for assay of A. californica nuclear polyhedrosis virus, but the sensitivity was lower than with S. frugiperda cells.     

In Vitro Survey of Autographa californica Nuclear Polyhedrosis Virus Interaction with Nontarget Vertebrate Host Cells

Thirty-five nontarget host cell lines, 23 of human and 12 of nonhuman vertebrate origin, were exposed to Autographa californica nuclear polyhedrosis virus preparations derived from four different sources: polyhedra, hemolymph, cell culture medium, and cultured cells. The virus and cells were incubated together at two different temperatures, 28 or 37°C, for four different lengths of time, 16, 40, 64, or 168 h, and the cells were assayed for the presence of virus by a peroxidase-antiperoxidase detection method. The estimated sensitivity of the assay as routinely conducted was 0.98 ng of alkali-liberated viral protein and 1.95 ng of budded viral protein per mm². No evidence of frank replication was obtained in any of the 35 cell lines tested, although virus uptake appeared to be quite common. Virus uptake was confirmed in some cases by electron microscopy. The degree of virus uptake appeared to be dependent on cell type, time and temperature of incubation, and viral phenotype. Virus purified from polyhedra was generally taken up more readily than were the other forms tested.     

Replication and infectivity of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, produced in cells grown in vitro

A virus isolated from the alfalfa looper, Autographa californica, replicated successfully and rapidly in a suspended ovarian cell line of the cabbage looper, Trichoplusia ni. Polyhedra were observed in the nucleus of cells within 20 hr after inoculation. The cytopathological changes typical of nuclear polyhedrosis infections were observed, and an average of 64 polyhedra/cell were produced. These polyhedra were quantitatively as infectious to cabbage looper larvae as those produced in vivo. In addition, they were infective to Heliothis virescens, Pectinophora gossypiella, Spodoptera exigua, A. californica, and Anagrapha falcifera.     

Two tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses

Two media supporting the growth of several established lepidopteran cell lines in monolayer and suspension culture are described. The medium designated $\text{BML-TC}\text{10}$ was developed specifically as an inexpensive medium for production of cells of Spodoptera frugiperda and the homologous nuclear polyhedrosis virus (NPV) of this species. Simultaneously, a second medium was formulated in which the amino acid requirements were provided by enzymatic protein hydrolysates, one of which was termed $\text{BML-TC}\text{7A}$. Several cell lines could be adapted easily to this medium. $\text{BML-TC}\text{10}$ supported growth of S. frugiperda cells and production of the NPV's of S. frugiperda and Autographa californica. $\text{BML-TC}\text{7A}$ supported the growth of cells of S. frugiperda. Carpocapsa pomonella, Heliothis zea, and Trichoplusia ni. Cells of the latter produced the polyhedra of T. ni and A. californica NPV's in this medium.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

A new variant of Autographa californica nuclear polyhedrosis virus

A variant of the baculovirus, Autographa californica nuclear polyhedrosis virus, has been isolated in Idaho during an epizootic disease in a field population of A. californica. Genotypic characterization indicates that the virus is distinct from variants previously characterized. Analysis of five clones, derived by plaque purification in cell culture, indicates relative homogeneity of the original virus isolate. Further exploration of the factors involved in natural genetic variability of baculoviruses is appropriate.     

Strategies to increase the use of entomopathogens

Synchronized cultures of the TN-368 insect cell line were infected with a nuclear polyhedrosis virus from the alfalfa looper, Autographa californica, during different phases of the cell cycle. Cultures exposed to virus during the middle and late S phase have higher percentages of infected cells than cultures inoculated with virus in the G₂ phase. The amount of virus produced from each infected cell (polyhedra and plaque forming units) is not significantly different between cultures infected at all phases of the cell cycle.     

Differential requirements of two insect cell lines for growth in serum-free medium

Summary The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 × 10⁶ TCID₅₀ extracellular virus and 4.4×10⁸ polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.     

Propagation of Autographa californica nuclear polyhedrosis virus in cell culture: Methods for infecting cells

Autographa californica nuclear polyhedrosis virus (AcNPV) produced in Trichoplusia ni (TN-368) cells was used to infect other cell cultures. Methods were developed to recover and obtain high titers of virus from infected cells for subsequent use as inocula. To release cell-associated nucleocapsids, the cells were lysed by sonication and freeze-thawing. The infectivity of enveloped nucleocapsids was greatly reduced by freeze-thawing, while sonication was not as detrimental. The titer of plaque-forming units (pfu) was reduced about 12-fold when passed through 0.45-μm filters. The virus and cells were manipulated to determine the most efficient methods for inoculating cells while yielding the highest numbers of polyhedra. The viral inocula may be left on cells during virus replication, and cells may be centrifuged at 380 g prior to exposure to virus without affecting the yield of polyhedra. The production of polyhedra is affected by cell density, and, of the densities tested, 7.65 × 10⁵ cells/ml yielded the maximum number of polyhedra per cell (142). However, the highest number of polyhedra per milliliter of culture (2.2 × 10⁸) was obtained with 3.8 × 10⁶ cells/ml. The numbers of polyhedra per cell did not vary when cells were taken from fermentor cultures at 0–144 hr and were infected with virus.     

Comparison of nuclear polyhedrosis virus replication in five lepidopteran cell lines

Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides.     

Characterization of a new insect cell line that is derived from the neonate larvae of Papilio xuthus (Lepidoptera: Papilionidae) and its susceptibility to AcNPV

The cell line RIRI-PX1 was established from neonate larval tissues of Papilio xuthus by performing primary cultures in the modified Grace medium that was supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of spindle-shaped and spherical cells which attached themselves to the flask. The population-doubling times (PDTs) at the 50th and 60th passage were 42.5 h and 42.1 h respectively. The average chromosome numbers of RIRI-PX1 cell line from passage 5 to passage 50 ranged from 103 to 199. It was confirmed that RIRI-PX1 cell line was derived from P. xuthus by comparing the mitochondrial cytochrome c oxidase subunit I gene (COI) of RIRI-PX1 cells and P. xuthus eggs. This cell line was susceptible to the Autographa californica nucleopolyhedrovirus (AcNPV) and produced high yield of polyhedral occlusion bodies (43.9 OBs/cell) after 10 days of infection by AcNPV. The virus titer of AcNPV infected RIRI-PX1 cells was 3.25 × 10⁷ TCID₅₀/ml. We concluded that the RIRI-PX1 cell line is established from the neonate larvae tissues successfully and the cells of the cell line are sensitive to AcNPV.     

Utilizing the macroporous packed bed for insect cell/baculovirus expression

Due to their high porosity and biocompatibility, polyurethane foam (PUF) and cellulose foam were adopted for insect cell immobilization and baculovirus expression. Spodoptera frugiperda (SF-21) cells were grown within the macroporous matrix and then infected by Autographa californica nuclear polyhedrosis virus (AcNPV) which was encoded with human interleukin-5 (hIL-5) gene. An appropriate initial cell loading density and medium circulation velocity determined from the previous study were applied in this actual cell cultivation experiments to obtain a uniform initial and final axial cell distribution. The growth of insect cells and the expression of baculovirus were successful in the macroporous packed bed systems used. The final average cell density in cellulose foam achieved was 5.2×10⁷?cells/cm³ and 4.3×10⁷?cells/cm³ in PUF. Under the conditions of sufficient nutrition and oxygen supplement, the average productivity of hIL-5 in cellulose foam packed bed bioreactor reached 7.2×10⁷ unit/l-day. With 50% fresh medium replacement after viral infection, the average productivity of hIL-5 in PUF packed bed reached 8.4×10⁷ unit/l-day, about two fold than that without any fresh medium replacement at infection.     

Convenient Desktop-scale Production of the Extracellular Domain of the Human Growth Hormone Receptor by an Insect-baculovirus Secretion System Using a Protein-free Culture

Expression of a gene encoding the extracellular domain of the human growth hormone receptor (hGHR-ED) inserted into the genome of Autographa californica nuclear polyhedrosis virus was done using a desktop-scale spinner culture. Spodoptera frugiperda 9 (Sf9) cells infected with the recombinant virus secreted a protein with hGH-binding activity into the medium. Oxygen supplementation was required for high level secretion of the product. The highest cell production capability was estimated at more than 15 mg hGHR-ED/liter of culture. A protein-free medium supported the production similar to that obtained in traditional serum-containing media. This spinner culture system is simple to operate, and does not require expert knowledge of culture techniques.     

Construction of Stably Transformed Bm5 and Sf9 Cells Displaying Green Fluorescence by Using Autographa californica Nuclear Polyhedrosis Virus IE1 Gene

Transformed Bm5 or Sf9 cells displaying green fluorescence were constructed by using Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early gene (ie 1). Green fluorescent protein (gfp) gene was introduced under the control of the AcNPV ie 1 promoter to yield expression plasmid pAcIE1-GFP. It was transfected into Sf9 or Bm5 cells and cell clones expressing GFP were selected by fluorescence microscopy. Genomic DNA from transformed cells was isolated and integration of AcNPV ie 1 gene harboring gfp gene was confirmed by PCR using AcNPV ie 1 gene-specific primers. The GFP was successfully expressed in the cytoplasm of insect cells transformed with pAcIEI-GFP and the expressed GFP was maintained during cell division. Furthermore, GFP expression by AcNPV ie 1 promoter in transformed cells was not interfered with viral replication. This suggests that transformed cells displaying foreign gene product by using AcNPV ie 1 promoter will be useful for the diverse applications of the insect cells.     

Protein production (β-galactosidase) from a baculovirus vector inSpodoptera frugiperda andTrichpolusia ni cells in suspension culture

Protein production capabilities ofTrichpolusia ni (TN 368) cells andSpodoptera frugiperda (Sf9) cells were compared in GTC100 medium in suspension culture using as a vector a genetically engineeredAutographa californica nuclear polyhedrosis virus. TN 368 produces more -galactosidase than Sf9, on a per cell basis (2.2×10⁵ and 1.7×10⁵ units/ 10⁶ cells¹ respectively). In growth experiments serum-free medium supported a higher maximum Sf9 cell density (4±1×10⁶ cells/ml) than the serum- based media (1.5±5×10⁶ cells/ml in GTC100 and 2±1×10⁶ cells/ml in TNM-FH). However, using a cell density of 5×0⁵ cells/ml, the productivity per cell varied, from a low of 4.5×10⁴ units in EX-CELL-400 medium to a high of 7.6×10⁴ units in TNM-FH. The TN 368 cells were twice a large as Sf9 cells and appeared to be more shear sensitive than Sf9 cells.     

Increased virulence of Autographa californica nuclear polyhedrosis virus by mutagenesis

A mutant of the Autographa californica nuclear polyhedrosis virus (AcMNPV) with increased virulence in Trichoplusia ni larvae was isolated following replication of a random virus clone in the presence of 2-aminopurine. The LT₅₀ of the mutant, designated HOB, was significantly shorter than those of either the wild isolate or parental clone of AcMNPV. Also, fifth-instar larvae infected with this mutant gained significantly less weight and consistently produced more virus occlusion bodies than larvae infected with the wild isolate or parental clone. No alterations in the in vitro replication of nonoccluded virions, occluded virus structural proteins, or DNA restriction endonuclease patterns were observed with the HOB mutant.