Heliothis Zea Nuclear Polyhedrosis Virus Research Articles

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 × 10 5 PFU/ml (AM1-A4), with the parental cell lines AM3 and AM1 producing 14.8 and 26.4 × 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 × 10 6 PIB/ml (AM1-B3), while the parental cell lines generated 6.5 (AM3) and 12.9 × 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.

Transfection of cloned Heliothis zea cell lines with the DNA genome of the Heliothis zea nuclear polyhedrosis virus

Transfection conditions were optimized for the cloned UND-K derivative of the IPLB-HZ 1075 cell line using the calcium-phosphate co-precipitation technique and the DNA genome of the Heliothis zea S-type nuclear polyhedrosis virus. Optimal efficiencies were obtained using supercoiled viral DNA, and by extending the adsorption period for the diluted precipitate to 12 h. Transfection efficiencies ranging from 0.5 to 1.3 × 10 3 plaque forming units per μg of supercoiled viral DNA were routinely obtained for UND-K cells and HzS-15 viral DNA. Transfection efficiencies were compared for 10 other cloned Heliothis cell strains and the uncloned parental IPLB-HZ 1075 cell line. The cloned cell strains UND-F, L, and U were incapable of transfection, while UND-I and G were 3 and 131 fold (respectively) less efficient than UND-K. The UND-K cells and the calcium phosphate transfection procedure permit relatively efficient in vitro manipulation of the Heliothis zea NPV virus genome.

Yield and activity of theHeliothis zea single nuclear polyhedrosis virus propagated in cloned and uncloned lines ofHeliothis cells

Heliothis cell lines originated from different laboratories were characterized by isoenzyme analysis and then evaluated for their ability to produce the single nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). A cloned cell line (designated Hzlb3), whose homogeneity was supported by both morphological and isoenzyme analysis, was derived from a parental line (Hzl). Significantly greater yields (about 10-fold) of tissue-culture-derived, non-occluded virus (TCNOV) were obtained when compared to the parental line. The Hzlb3 clone also gave significantly higher yields of TCNOV than the Hz3 and UND-K cell lines. Although lines Hzl, Hz3, and Hzlb3 produced significantly more polyhedral inclusion bodies (PIB) than line UND-K, the infectivity of PIB from UND-K equaled that of lines Hzl and Hzlb3.

Characterization of clonal populations of theHeliothis zea cell line IPLB-HZ 1075

A total of 24 clones (HZ 1075/UND-A through X) were initially isolated by dilution plating from the established IPLB-HZ 1075 cell line. Many of the isolates with highly vacuolated cytoplasms eventually died during subculturing. The cloned cell strains differed in their predominant morphology, cell doubling times, and relative ability to support replication of the singly encapsulated nuclear polyhedrosis virus ofHeliothis zea (HzSNPV). The origin of the cloned cell strains was confirmed by comparing their isozyme profiles with those of the parental IPLB-HZ 1075 cell line andH. zea larvae using stains for fumerate hydratase, lactate dehydrogenase (LDH), and malic dehydrogenase (MDH). One dipteran and several lepidopteran cell lines maintained in our lab were also separable using stains for LDH and MDH.

Replication of Heliothis zea Nuclear Polyhedrosis Virus in Insect Cell Culture: Virus-induced and Structural Proteins

Summary The replication of Heliothis zea nuclear polyhedrosis virus was studied in the H. zea (IPLB-HZ-1075A) insect cell line. Infectious extracellular virus was first detected at 24 h post-infection (p.i.) and reached a maximum titre of 1 × 107 p.f.u./ml at 90 h p.i. Viral DNA synthesis was detected by 14 h p.i. and increased exponentially until 32 h p.i. Using [35S]methionine pulse-labelling, 38 virus-induced proteins were identified, ranging in mol. wt. from 13K to 128K. Virus-induced protein synthesis was temporally regulated, with new proteins detected between 8 and 28 h p.i. Host cell protein synthesis continued throughout the virus replication cycle (48 h), although at gradually decreasing rates. Fifteen extracellular virus structural proteins were identified, and all but one comigrated with virus-induced proteins. Thirty-two structural proteins of tissue culture-derived, occluded virus were identified using silver staining procedures.

Monoclonal antibodies identify conserved epitopes on the polyhedrin of Heliothis zea nuclear polyhedrosis virus

Monoclonal antibodies were formed against polyhedrin from the Heliothis zea S nuclear polyhedrosis virus. The cross-reactivity of these antibodies with polyhedrins and granulins of other occluded baculoviruses indicates that group and subgroup epitopes exist. Evidence is presented that multiple copies of one epitope or a series of closely related epitopes exist on the polyhedrin molecule. The monoclonal antibodies, with the exception of one IgM-producing hybridoma clone, were all of the IgG1 isotype. Clones producing antibodies with identical patterns of reactivity with heterologous polyhedrins and/or granulins but exhibiting different affinities for the homologous antigen (HzSNPV polyhedrin) are demonstrated.

The detection of FP plaque variants of Heliothis zea nuclear polyhedrosis virus grown in the IPLB-HZ 1075 insect cell line

The detection of FP plaque variants of Heliothis zea nuclear polyhedrosis virus grown in the IPLB-HZ 1075 insect cell line

A Physical Map for the Heliothis zea SNPV Genome

SUMMARY Restriction sites on the Heliothis zea nuclear polyhedrosis virus (HzSNPV) genome were mapped for the endonucleases BamHI, EcoRI, HindIII, SstI, XhoI and KpnI. XhoI-F was used as the zero-point of the physical map because it hybridized to the HindIII-V fragment which codes for the 3′ end of the polyhedrin gene of the Autographa californica nuclear polyhedrosis virus (AcMNPV). The DNA homology between eight AcMNPV cloned fragments and HzSNPV DNA was mapped using Southern hybridization and the locations of HzSNPV DNA sequences sharing homology with AcMNPV gene sequences are discussed.

Effect of Spray Adjuvants on Heliothis zea (Lepidoptera: Noctuidae) Nuclear Polyhedrosis Virus Efficacy1

Certain characteristics of Elcar ( Heliothis zea nuclear polyhedrosis virus) spray adjuvant treatments were determined in an effort to explain or identify properties of the combination which influence efficacy of the virus against Heliothis spp. on cotton. Neither Coax nor Gustol (adjuvants)affected the dosage mortality response of larvae of H. zea (Boddie) to Elcar. Laboratory studies also failed to detect ultraviolet screening properties of the adjuvant sat the rate tested (2.5% concentration). Coax and Gustol appeared to primarily affect efficacy by altering feeding or spray characteristics. Third-stage H. zea larvae consumed more cotton leaf discs treated with the virus when an adjuvant was included in the treatment. Correlation analysis showed adjuvant rate to be positively related (r = 0.92) to larval mortality, although percent concentration of adjuvant appeared to be more important than rate in influencing the spray characteristics of Elcar treatments.

Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line

This paper describes the replication and serial passage of Heliothis zea nuclear polyhedrosis virus (NPV) in a H. zea cell line. It was demonstrated that long-term serial passages of the H. zea NPV in homologous host cell culture decreased both the total number of polyhedral inclusion bodies (PIBs) produced and the infectivity of the supernatant as measured by TCID 50. The growth curve indicated that infectious material was released from cells 24 hr postinfection (p.i.) and approached a maximal titer 3 days p.i. The kinetics of H. zea NPV decay at 4°, 27°, and 37°C were determined. Infectivity was not detected after 3 weeks at 37°C, but approximately 10 3.5 TCID 50/ml activity was still present after 3 and 8 weeks storage at 27° and 4°C, respectively. Electron microscopy confirmed the presence of single embedded virions in the inoculated cells.

Replication of Heliothis zea baculovirus in an insect cell line.

The baculovirus, Heliothis zea nuclear polyhedrosis virus (HzSNPV), was grown in the H. zea cell line, IPLB-1075. Light microscopy observations showed that 95% of inoculated cells had CPE by 6 days postinoculation (p.i.). Growth curves of intracellular and extracellular virus showed that maximum titers (TCID50 units) were reached 3-4 days p.i. Studies on HzSNPV morphogenesis revealed that ultrastructural events were typical of baculovirus replication in vitro. EcoRI restriction enzyme analyses of viral DNA produced in larvae and in cell culture were identical and indicated that this virus was HzSNPV.

Comparative peptide mapping of baculovirus polyhedrins

Amino terminals and two-dimensional high-voltage peptide maps of tryptic digests of polyhedrins from Heliothis armigera nuclear polyhedrosis virus (NPV), Heliothis zea NPV, and Anticarsa gemmatalis NPV were compared with previously characterized granulins and polyhedrins. Similarities and differences were detected in the tryptic maps, while each protein produced a unique peptide map composite. Amino-terminal determination of eight polyhedrins and granulins resulted in three different end groups.

Long-term feeding and other toxicity-pathogenicity studies on rats using a commercial preparation of the nuclear-polyhedrosis virus of Heliothis zea

A commercial preparation of the nuclear-polyhedrosis virus of Heliothis zea was administered orally and parenterally to rats to evaluate its carcinogenicity. The data collected in this study indicated no numerically significant difference in the incidence of neoplasia in the treated groups when compared to the controls.

Inhalation and dermal allergenicity studies of a nuclear-polyhedrosis virus of Heliothis zea in guinea pigs

Guinea pigs were exposed to a commerical preparation of the nuclear-polyhedrosis virus of Heliothis zea (Biotrol VHZ 2 2 Manufactured by Nutrilite Products, Inc., Buena Park, California 90620. ). The material was administered in the form of polyhedral inclusion bodies (PIB) and free virus rods by inhalation and intradermal injection. In all tests, there were no discernible differences between treated and control groups.

Toxicity-pathogenicity studies of a nuclear-polyhedrosis virus of Heliothis zea in white mice

A commercial preparation containing polyhedral inclusion bodies (PIB) of the nuclear-polyhedrosis virus of Heliothis zea (Biotrol VHZ2

Dosage-mortality data on a nuclear-polyhedrosis virus of the bollworm, Heliothis zea

The need for a reliable method of estimating the virulence (potency) of a nuclear-polyhedrosis virus of the bollworm, Heliothis zea, is discussed. Dosage-mortality data is obtained from a simple reproducible bioassay technique. Early third-instar larvae, averaging 20 mg in weight, were used throughout this study; the larvae were held at 28° ± 2°C throughout the testing period. The calculated ld 50 values in terms of polyhedral inclusion bodies (PIB) per larva for three different Heliothis viruses (Biotrol ®, Sudan, and Ivory Coast) were 269, 344, and 459, respectively. Laboratory data show that Biotrol ® Heliothis zea nuclear-polyhedrosis virus is more virulent than the other two viruses when tested on the larvae of Heliothis zea.

The effect of human gastric juice on the pathogenicity of Heliothis zea nuclear-polyhedrosis virus

This paper describes the effects of time exposure to human gastric juice on the pathogenicity of polyhedral inclusion bodies (PIB) containing the nuclear-polyhedrosis virus of the corn earworm, Heliothis zea. PIB, after purification, were incubated in vitro at 37°C for time exposures of 10, 20, 40, 60, 90, and 120 min with human gastric juice at pH 2.1 and with human gastric juice neutralized to pH 7.0. After exposure the PIB were bioassayed against 15–25 mg larvae of H. zea. Results showed that incubation at pH 2.1 inactivated the PIB very rapidly (50%, 40 min; 100%, 120 min), whereas incubation at pH 7.9 had little effect on pathogenicity.

Human feeding tests using a nuclear-polyhedrosis virus of Heliothis zea

A preparation containing live virus from the cotton bollworm, Heliothis zea, killed by the specific nuclear-polyhedrosis virus was fed to 10 men and women who volunteered as test subjects. Six other men and women (controls) were fed insect protein separated from the same preparation of polyhedrosis virus. Over the course of 5 days, each test subject consumed 5.82 billion polyhedra. Complete physical examinations, including a variety of laboratory tests of all test subjects and controls, made immediately before the test began and 10 and 30 days after the feeding showed no significant change in the condition in any individual in the test or control groups and no differences between the test subjects and controls.

The nuclear-polyhedrosis virus of Heliothis zea (Boddie) and Heliothis virescens (Fabricius) : V. Toxicity-pathogenicity of virus to white mice and guinea pigs

White mice and guinea pigs were exposed to the nuclear-polyhedrosis virus of Heliothis zea (Boddie) and Heliothis virescens (Fabricius). The virus was administered in the form of polyhedral inclusion bodies, free virus rods, or polyhedral protein by inhalation, per os feeding, or intravenous, intradermal, intraperitoneal, and intracerebral injections. One death occurred, caused by an acute pneumonia, in 155 virus-treated and 75 nonvirus-treated animals. The animals showed normal weight gains and no abnormalities or pathological conditions were detected.

The nuclear-polyhedrosis virus of Heliothis zea (Boddie) and Heliothis virescens (Fabricius) : I. Virus propagation and its virulence

Results of a pilot program demonstrated that (1) The nuclear-polyhedrosis virus of Heliothis zea (Boddie) and Heliothis virescens (Fabricius) can be propagated in larvae reared on a semisynthetic diet; (2) the virus thus produced is effective against both species; (3) the techniques are applicable to commercial production and costs may be in the realm of commercial feasibility.