Biosafety of Recombinant and Wild Type Nucleopolyhedroviruses as Bioinsecticides

Abstract

The entomopathogenic Autographa californica (Speyer) nucleopolyhedrovirus (AcMNPV) has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT) as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV) were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 × 1012 PIBs/feddan, feddan = 4,200 m2) and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field), AcMNPV, and SlNPV) using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 μg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%), AcAaIT-field (1.2%), and SlNPV (4.0%). Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female) that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal injection at doses of 1 × 108 or 1 × 107 PIBs/rat, respectively) appeared to be healthy and showed increased body weight at 21 days posttreatment. The effect of virus administration on hematological, serum biochemical, and histopathological parameters were determined. Slight to moderate differences were observed in most of the hematological parameters. Specifically, serum proteins were decreased markedly in female rats treated orally with SlNPV, and in male rats injected with AcAaIT. SDS-PAGE analysis also showed some changes in serum protein profiles. No marked changes in acetylcholine esterase (AChE) activity were found. Changes in serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, creatinin, and urea were also observed. Immunohistochemical observation of tissues from stomach, intestine, liver, kidney, brain, spleen, and lung also showed slight changes. Fish (Tilapia nilotica) were also exposed to AcAaIT, AcMNPV or SlNPV by incorporating each of the viruses into diet (1 × 109 PIBs/group). No mortality...

Regulation analysis of AcMNPV-mediated expression of a Chinese scorpion neurotoxin under the IE1, P10 and PH promoter in vivo and its use as a potential bio-insecticide

“…Recombinant DNA technology has been used to overcome this and other potential drawbacks that have been associated with the largescale use of baculovirus-based biopesticides (Kamita et al 2005;Inceoglu et al 2006). A major strategy for improving the insecticidal efficacy of the baculovirus has been the insertion of insect-selective neurotoxin genes into the baculovirus genome (Ashour et al 2007). Many studies have been carried out to assess the expression of foreign gene under different promoters.…”

An Introduction to Entomopathogenic Microorganisms

“…The baculovirus-based bioinsecticides are host specific and safe to environment, human and animal health with no toxic residues and can be combined with other strategies for IPM (Monobrullah 2003;Ashour et al 2007). Detailed classification, morphology and use of baculoviruses for control of lepidopteran pests are given under Chap.…”

Recent Advances in Our Knowledge of Baculovirus Molecular Biology and Its Relevance for the Registration of Baculovirus-Based Products for Insect Pest Population Control

“…mice, rats, rabbits) in vivo as well as in mammalian cell cultures. Baculovirus active ingredients and end products have been ingested and inhaled by, injected (intravenous, intraperitoneal, intramuscular) into, and applied to the skin and eyes of test animals without detrimental effects that could be attributed to the baculovirus tested (Table 1) (Ashour et al, 2007;Gröner, 1986;Ignoffo, 1975;Lightner et al, 1973). Many species of baculoviruses have been tested on numerous species of animals at doses that are many times those that could be acquired in the field.…”

“…(from Gröner, 1986;Ignoffo, 1975). a, b, c, d: Data obtained from Lightner et al (1973), Ashour et al (2007), and Health Canada PMRA Regulatory Notes REG2000-10 and RD2009-05, respectively.…”