Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures

The aim of our study was to establish an efficient system for thein vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.Abbreviations Ac-NPV Autographa californica Nuclear Polyhedrosis Virus - BV Baculovirus - MOI Multiplicity Of Infection - ECV Extracellular Virus     

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.     

Serological relationships of five nuclear polyhedrosis viruses from lepidopterous species

The serological relationships of five nuclear polyhedrosis viruses (NPV) were investigated using the immunodiffusion technique with intragel absorption. Reciprocal tests demonstrated that virion fractions from Autographa californica multiple embedded virus (MEV), Heliothis armigera MEV, and H. zea single embedded virus (SEV) are not related to each other or to virions from Trichoplusia ni SEV and Pseudoplusia includens SEV. Virion fractions of T. ni and P. includens NPV were shown to be closely related, sharing several antigens. Matrix fractions possessed a common group antigen and one or two antigens specific for the individual NPV with the exception that T. ni and P. includens NPV shared one of these antigens. The specific antigens of the matrix fraction were also shared with the homologous virion fraction.     

Conservation of genome organization in two multicapsid nuclear polyhedrosis viruses.

The genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata was mapped by examining overlapping HindIII fragments from cosmid clones which had been constructed from partial HindIII digests of viral DNA. Five OpMNPV cosmid clones containing fragments encompassing the entire OpMNPV genome were hydridized to blots of DNA from the multicapsid nuclear polyhedrosis virus of Autographa californica. The hybridization pattern indicated that the genomes of these viruses are similarly organized.     

Comparative biochemical studies of polyhedral proteins of nuclear polyhedrosis viruses]

Using disc polyacrylamide gel electrophoresis, the molecular weights of polyhedral proteins of nuclear polyhedrosis viruses (NPV) of Porthetria dispar, Mamestra brassicae, and Aporia crataegi were found to be 28000 +/- 3000. It was shown that NPV polyhedra of Bombyx mori, Galleria mellonella, P. dispar, and M. brassicae contain a protease. During dissolution of the polyhedra at pH 10,5 this protease specifically cleaves the matrix protein into 2--5 fragments. The amino acid compositions of NPV polyhedral proteins of P. dispar, M. brassicae, A. crataegi, Hyphantria cunae were shown to be very similar. It was found that tyrosine is a C-terminal amino acid of NPV polyhedral proteins of P. dispar, M. brassicae, and A. crataegi.     

Interaction of vertebrate viruses and insect nuclear polyhedrosis viruses with transplantable diploid embryonal drosophila cells

When the Drosophila cells were infected with the mixo- and arboviruses, in case of influenza A/WSN virus a rise in the titre and slight cytopathogenic effect with the subsequent decrease in the titre was observed. Since the decrease in the virus titer was not observed when actinomycin D was added, it was supposed that interferonlike inhibitor may be produced by the infected cells. Vacuolization and increase in the size of the infected cells were caused by all the nuclear polyhedrosis viruses tested. The number of the infected cells depended on the virus type and multiplicity of the infection.     

Characterization of DNA from three nuclear polyhedrosis viruses pathogenic for Choristoneura sp

Three nuclear polyhedrosis viruses isolated from larvae of the insect genus Choristoneura showed polyhedrins of 28–30,000 daltons, genome sizes of 78–82 × 10⁶ daltons, and guanine plus cytosine contents of 47.9–49.4%. It was demonstrated by comparison of restriction endonuclease fragment patterns that two of the viruses are closely related genetically.     

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.     

Genotypic variation among wild isolates of two nuclear polyhedrosis viruses isolated from Spodoptera littoralis

Diseased Spodoptera littoralis larvae were collected from 21 different regions of Israel. Nuclear polyhedrosis viruses were isolated from these larvae, and viral DNAs were compared by restriction endonuclease analysis. Two distinct virus types, represented with approximately equal frequency, were found. Several wild isolates of each virus type exhibited minor restriction pattern differences. Plaque purification of the wild isolates revealed the presence of additional genotypic variants.     

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.     

Serological relationship of nuclear polyhedrosis viruses: I. Hemagglutination by polyhedral inclusion body protein from the nuclear polyhedrosis virus of Porthetria (= Lymantria) dispar

Polyhedral inclusion body protein (PIBP), harvested from the nuclear polyhedrosis virus (NPV) infecting the gypsy moth, Porthetria dispar, illicits the hemagglutination of chicken erythrocytes. Antisera to PIBP, polyhedral inclusion bodies (PIB), and virions (RODS) from the NPV's infecting P. dispar and the European Pine Sawfly, Neodiprion sertifer, inhibits hemagglutination when utilized to neutralize the PIBP from P. dispar. The crossreactivity of antisera to viral components from N. sertifer, a hymenopteran insect, with viral antigens from P. dispar, a lepidopteran insect, demonstrates a serological relationship exists between two viruses which have widely separated host ranges.     

Replication and passage of alfalfa looper nuclear polyhedrosis virus plaque variants in cloned cell cultures and larval stages of four host species

As alfalfa looper nuclear polyhedrosis virus (NPV) was serially passed through TN-368 cell cultures, the percentage of the cell population that developed infection with the MP variant decreased, while cells infected with FP variant increased. The MP variant was more virulent to Trichoplusia ni than the FP variant. Cells from the TN-368 cell line and strains derived from single cells were infected with isolated MP plaques. The viral progeny remained homogenous after one or occasionally two passages in cultured TN-368 cells. However, further serial passes in TN-368 cells or one pass in cell strains resulted in loss of the homogeneity, and the FP variant was detected. Four species of Lepidopterous larvae were also infected with the MP variant. Both variants were detected in viral progeny from diseased larvae.     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.