Comparison of biophysical and morphological properties of occluded and extracellular nonoccluded baculovirus from in vivo and in vitro host systems.

Electron microscopic examination and buoyant density profiles of nonoccluded Rachiplusia ou and Autographa californica nuclear polyhedrosis viruses purified from both infectious insect hemolymph and cell culture medium revealed that the viruses are enveloped, single nucleocapsids. The envelopes exhibited variation in the amount and degree of fit with regard to the nucleocapsids. This was determined by: (i) electron microscopic observations of virus budding from the surface of infected cells; (ii) electron microscopic observations of negatively stained preparations of pelleted, highly purified, nonoccluded enveloped particles; and (iii) the resolution and density distributions of nonoccluded virus in sucrose gradients after centrifugation to equilibrium; all were compared with virus extracted from polyhedra. Peplomers, ovserved on the surface of enveloped nucleocapsids of nonoccluded virus, are not associated with polyhedra-derived virus. Density gradient analysis indicated that virus from insect hemolymph and culture medium exhibited similar densities of approximately 1.17 to 1.18 g/ml. This is significantly different from the buoyant density of an alkali-liberated, enveloped single nucleocapsid (1.20 g/ml). Results of this study show that the nonoccluded forms of two nuclear polyhedrosis viruses from two different sources, hemolymph and cell culture, are similar with regard to several morphological and biophysical characteristics but are quite different from the alkali-liberated, polyhedra-derived form of the virus.     

Strain selection during serial passage of Trichoplusia in nuclear polyhedrosis virus.

Two strains of a nuclear polyhedrosis virus (NPV) of Trichoplusia ni were isolated on the basis of plaque morphology. They are designated as MP (having greater than 30 polyhedra per nucleus) and FP (having fewer than 10 polyhedra per nucleus). Serial, undiluted passage of plaque, purified MP nonoccluded. Virus (NOV) in tissue culture led to the production of the FP phenotype detectable at passage 9. With continued serial, undiluted passage, FP became the predominant strain. Comparative growth curves showed that FP NOV are released faster than MP NOV. MP morphology was not observed after 14 serial, undiluted passages of plaque-purified FP. By the plaque neutralization assay, NOV from both strains of virus was neutralized by the homologus and heterologous antisera. The FP phenotype was observed when FP virus was grown in culture at 17, 22, and 27 C. Hence, the FP phenotype was not considered to be the result of temperature-inhibited crystallization of polyhedrin under standard tissue culture conditions. The NOV of both strains killed insects when injected directly into the hemocoele of T. ni larvae. Only MP inclusion bodies were virulent per os. The FP inclusion bodies fed to cabbage looper larvae did not kill, and no infectious agent could be detected in the hemolymph. Electron micrographs of MP polyhedra showed bundles of nucleocapsids of normal length within the polyhedra, whereas FP polyhedra contained heterogeneous, electron-dense material, which could account for their lack of pathogenicity.     

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.     

Functional differences between occluded and nonoccluded viruses of a nuclear polyhedrosis of the silkworm, Bombyx mori.

Comparative infectivity and virus neutralization studies on occluded and nonoccluded viruses of Bombyx mori nuclear polyhedrosis revealed that the infectious unit causing peroral infection differed from that causing hemocoelic infection. There were functional differences between the occluded (mainly virons with envelopes) and the nonoccluded virus (mainly virions without envelopes) preparations. The peroral infection was largely due to the virion with an envelope (peroral infectious unit), and the hemocoelic infection was due largely to the virion without an envelope (hemocoelic infectious unit). The apparent change of the virions with envelope to those without envelopes was detected as a slight increase in hemocoelic infectivity when the occluded virus was diluted and incubated at 4°C for more than 6 days.     

Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture

When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.     

Effect of light energy on alkali-released virions from Anagrapha falcifera nucleopolyhedrovirus

We compared the insecticidal activities of occluded and nonoccluded AfMNPV baculovirus obtained by dissolving the occlusion bodies (OB) with sodium carbonate. Droplet feeding and cotton leaf feeding bioassay techniques were used to determine the dose response against neonate Trichoplusia ni (Hübner) and loss of insecticidal activity when the virus was exposed to simulated sunlight from a xenon light source. Using droplet bioassays to determine a dose response, nonoccluded virus (NOV) was 20 times more active (LC(50) = 4.8 x 10(3) OB/ml, dissolved) than occluded virus (LC(50) = 9.6 x 10(4) OB/ml) when the samples remained wet. However, NOV lost activity when air dried before being tested by droplet (LC(50) > 1.0 x 10(6) OB/ml) or leaf feeding (LC(50) > 3.0 x 10(6) OB/ml) bioassays. Adding sucrose to NOV prevented the loss of insecticidal activity when samples were dried. The activity of NOV with 2% sucrose was similar to that of occluded virus samples, with or without sucrose, in both droplet feeding and leaf feeding assays. These results indicate that the OB protected the insecticidal activity of virions from the detrimental effects of drying. The OB also provided some protection from the detrimental effects of simulated sunlight (xenon) exposure. NOV samples exposed to xenon light had significantly greater loss of insecticidal activity than did similar samples of occluded virus. Without advancement in technologies, such as formulations, possible benefits of increased insecticidal activity from the use of nonoccluded virus is probably not sufficient to offset the rapid loss of activity due to drying or light exposure.     

Replication of a Nuclear Polyhedrosis Virus in a Continuous Cell Culture of Spodoptera frugiperda: Purification, Assay of Infectivity, and Growth Characteristics of the Virus

Nonoccluded virus, polyhedra, and occluded virus were purified from a continuous cell culture of Spodopera frugiperda infected with nuclear polyhedrosis virus. The optimal temperature for the replication and lateral transmission of infectivity for the nuclear polyhedrosis viruses (NPV) in cell culture was 27 C. End-point dilution and plaque assay procedures for the measurement of infectivity are described and compared. Dose-response data demonstrated that a single particle could initiate an infection, and the validity of the relationship of 0.7 PFU per mean tissue culture infective dose (TCID(5 0)) further substantiated the accuracy of these infectivity assays. Particle-infectious unit calculations gave a ratio of 62 to 310 nonoccluded virus particles TCID(5 0). Growth cycle and lateral transmission experiments indicated that infectious material was released from cells 12 h postinfection (p.i.) and approached a maximal titer 4 days p.i. The number of polyhedra, nonoccluded virions, and TCID(5 0) produced per cell was also presented. Typical yields of NPV produced per liter flask suggested that insect cell culture systems represent a feasible means by which the replication of these viruses could be investigated.     

Isolation, Complementation, and Initial Characterization of Temperature-Sensitive Mutants of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Sixteen temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were isolated. Several interesting phenotypes were observed. A large proportion of the mutants were unable to form polyhedral occlusion bodies (polyhedra) at the nonpermissive temperature (32.5°C). At 32.5°C, one mutant formed plaques in which the cells lacked polyhedra. Another mutant type was defective in the production of progeny extracellular nonoccluded virus and produced a “plaque” consisting of only a single cell containing polyhedra at 32.5°C. One mutant was defective in plaque formation, progeny nonoccluded virus formation, and polyhedra formation at 32.5°C. Several mutants produced nonoccluded virus but failed to produce plaques or polyhedra at 32.5°C. Other phenotypes were also distinguished. Complementation analyses, performed by either measuring the increase in extracellular nonoccluded virus formation or by observing polyhedra formation in mixed infections at 32.5°C, indicated the presence of 15 complementation groups. A high frequency of recombination was observed. Four of the mutants were found to be host dependent in their temperature sensitivity for polyhedra formation.     

The proteins of nonoccluded Autographa californica nuclear polyhedrosis virus produced in an established cell line of Spodoptera frugiperda

Nonoccluded virions have been isolated from the extracellular fluid of cultured Spodoptera frugiperda cells that have been infected with Autographa californica nuclear polyhedrosis virus. By sucrose density gradient centrifugation, infectious virus particles have been obtained that exhibit densities of about 1.19 g/cm³. Using staining and autoradiographic techniques, about 35 polypeptides, including some high molecular weight proteins, have been detected by analysis of the virion proteins on sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE). Two major proteins of MW 65,000 and 42,000 have been found.     

Baculovirus defective interfering particles are responsible for variations in recombinant protein production as a function of multiplicity of infection

Summary Defective interfering particles, present within a high passage inoculum ofAutographa californica nuclear polyhedrosis virus (AcMNPV), interfered with recombinant -galactosidase and infectious virus production in three insect cell lines. These particles were Selected during serial passage, were missing large parts of the AcMNPV genome, and caused large reductions in -galactosidase production at multiplicities of infection above 0.01 pfu/cell.     

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).     

A continuous process for the production of baculovirus using insect-cell cultures

Summary A continuous culture of insect cells (Spodoptera frugiperda) was used for continuous production of baculovirus (nuclear polyhedrosis virus fromAutographa californica). The system consisted of a cascade of two continuous stirred tank reactors (CSTRs). In CSTR I the insect cells were grown in suspension. This suspension was fed continuously to CSTR II where the virus infection occurred. For a period of about 25 days the average volumetric productivity was about 10⁷ polyhedra (virus particles occluded in protein capsules) and 10⁸ infectious NOVs (non-occluded virus particles) per cm³ effluent. This is equivalent to 25 polyhedra and 250 NOVs per infected cell, respectively. In one case, the percentage of infected cells was 65%, which is close to the theoretical value of 68%. After a run-time of 32 days a decrease of process productivity was observed, probably due to the so-called passage effect, a degeneration of the virus DNA.     

Continuous production of baculovirus in a cascade of insect-cell reactors

Summary Insect cells (Spodoptera frugiperda) were cultured in a continuous stirred-tank reactor. The effluent was led to a cascade of another two reactors, each containing half the volume of the cell-growth reactor, where the cells were infected with Autographa californica nuclear polyhedrosis virus. For about 10 days production of 10⁷ polyhedra (virus particles embedded in a protein capsule) per cm³ was achieved. This short production time compared to previous experiments involving an analogous system with a single infection vessel of equal volume to the cell-growth vessel is ascribed to the accelerated occurence of the so-called passage effect (a decrease of infectious virus with time). From the results of a computer model it was concluded that this passage effect was accelerated by the change in residence time distribution as compared to the earlier experiments. time distribution as compared to the earlier experiments. Offprint requests to: F. L. J. van Lier     

Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.     

Enhancement in activity of homologous and heterologous baculoviruses infectious to beet armyworm (Lepidoptera: Noctuidae) by an optical brightener

The nuclear polyhedrosis virus (NPV) from the beet armyworm, Spodoptera exigua (Hübner) (SeMNPV), was the most active virus tested against the beet armyworm (LC50 = 4.1 PIBs/mm2), followed by nuclear polyhedrosis viruses from the alfalfa looper, Autographa californica (Speyer) (AcMNPV; LC50 = 92.6 PIBs/mm2), and the celery looper, Anagrapha falcifera (Kirby) (AfMNPV; LC50 = 195.7 PIBs/mm2). In the case of the nuclear polyhedrosis virus from the bollworm, Helicoverpa armigera (Hübner), LC50s could only be obtained for five/six replicates, whereas LC50s could only be obtained for two/six replicates for the nuclear polyhedrosis virus from the wax moth, Galleria mellonella (L.) (GmMNPV). When an optical brightener Tinopal LPW was added to virus suspensions, LC50 values were reduced by 130-fold for both SeMNPV and AcMNPV and by 300-fold for AfMNPV. The addition of Tinopal LPW greatly increased the activities of HaMNPV and GmMNPV. In terms of speed of kill, Tinopal LPW reduced the LT50s for all nuclear polyhedrosis viruses by 30-40%.     

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.     

Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.

Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspects of substrate utilization and energy requirement during molluscan phagocytosis.

When certain ingredients were eliminated from a medium used to culture a cabbage looper cell line that can support replication of Autographa californica nuclear polyhedrosis virus, cells grew successfully and could be serially transferred a minimum of 44 times. Also, they maintained their ability to support replication of the Autographa californica virus, and the polyhedra produced were as infectious as those from cells grown on the original medium. The cost of the least expensive medium that would support cell growth was 2.8 times less than the cost of normal growth medium.     

Identification and characterization of a baculovirus occlusion body glycoprotein which resembles spheroidin, an entomopoxvirus protein.

A 37-kDa polypeptide specified by Autographa californica nuclear polyhedrosis virus was found to share significant homology with Choristoneura biennis entomopoxyvirus spheroidin protein, which is the major component of entomopoxvirus occlusion bodies. Antibodies raised against spheroidin cross-reacted with the 37-kDa protein and confirmed its expression in the late phase of wild-type baculovirus infection. Immunoblot analysis and fluorescence microscopy demonstrated that the protein was associated with purified A. californica nuclear polyhedrosis virus occlusion bodies and was absent in purified virions. Immunofluorescence studies localized the protein to the periphery of occlusion bodies and the internal membranes of cells infected with wild-type baculovirus. The open reading frame encoding this spheroidinlike protein was inserted into a baculovirus expression vector, and recombinant protein was synthesized under control of the polyhedrin promoter. Studies of the recombinant protein demonstrated that it was heterogeneous in molecular mass as a result of N-linked glycosylation. Tunicamycin inhibited carbohydrate addition and yielded proteins of 34 and 33 kDa.