Baculoviruses

Viruses within the family Baculoviridae contain large chains (80 a 130 kbp) of circular dsDNA, within a rod-shaped viral particle. Virions are occluded within occlusion bodies (OBs) as shown in Figure 8.4, known either as polyhedra or granules, constituted by a protein (polyhedrin or granulin).63 There are two genera within the family: Nucleopolyhedrovirus, typically known as nuclear polyhedrosis viruses or NPVs; and Granulovirus, typically known as granulosis viruses or GVs. NPVs are further divided into single nuclear polyhedrosis viruses (or SNPVs) and multiple nuclear polyhedrosis viruses (or MNPVs), depending on the number of nucleocapsids within each envelope. NPVs replicate only in the nuclei of infected cells, their OBs may be as large as 15 mm in diameter and contain many enveloped virions. GVs, on the other hand, contain only a singly enveloped nucleocapsid per granular OB, and therefore they are smaller than the NPV OBs (0.2 to 0.5 mm).

Some years ago, the so-called non-occluded viruses (NOV) were also included within the Baculoviridae family, but the International Committe on

Figure 8.4 Baculoviruses occlusion bodies (OB) isolated from Autographa californica.

The virus was originally isolated from the alfalfa looper. The OBs are composed of a protein matrix (polyhedrin for the nucleopolyhedrovirus, or granulin for the granulovirus) and are responsible for the primary infection of the host.

Figure 8.4 Baculoviruses occlusion bodies (OB) isolated from Autographa californica.

The virus was originally isolated from the alfalfa looper. The OBs are composed of a protein matrix (polyhedrin for the nucleopolyhedrovirus, or granulin for the granulovirus) and are responsible for the primary infection of the host.

Taxonomy of Viruses (ICTV) recently decided to take them out and create the new genus Nudivirus.64 This is a group of rod-shaped virions which are not occluded within an OB. The type-species of Nudivirus attacks the palm scarab, Oryctes rhinocerus, and constitutes an excellent example of the use of a virus with the classical biological control technique.

Some baculovirus genomes have been modified in order to obtain viral strains with foremost insecticidal properties, especially of AcNPV, BmNPV and HearSNPV.65 For this purpose, hormones, enzymes and insecticidal genes have been used to modify baculovirus genomes.66 With regard to the use of insecti-cidal toxins, a toxin gene Belt, encoding insectotoxin-1, obtained from the scorpion Buthus eupeus, was tested but the recombinant AcNPV obtained had no effect on either larval paralysis or on larval death.67 In contrast, genes coding for three arthropod venoms have been introduced into the AcNPV genome. One is the neurotoxin AaHIT from the scorpion Androctonus australis, which causes paralysis and death to a great variety of insects. Recombinant AcNPV virions expressing this toxin caused a 25% reduction in the median lethal time (LT50).68 Another recombinant organism with this AaHIT gene was obtained, but introduced into the genome of Helicoverpa armigera SNPV, with a reduction of 1734% in the median survival time (ST50).69 When recombinant viruses carrying Pyemotes tritici neurotoxin TxP-1 were used to infect greater wax moth larvae, Galleria mellonella (Lepidoptera: Pyralidae), paralysis was observed two days after infection. However, mortality was caused by the viral infection rather than by the toxin, with no significant difference with the parental viral strain.70

On the other hand, some toxins of animal origin were integrated into the AcNPV genome.71 These were: the m-Aga-IV venom from the spider, Agelenopsis aperta, and the As II and Sh1 toxins from the sea anemones, Anemonia sulcata and Stichodactyla helianthus. All three toxins show neurotoxic effects on insects. The efficiency of each recombinant virus varied both from each other and according to the insect species tested. The first toxin showed the highest activity on S. frugiperda larvae, with a 37% reduction in its lethal time to kill 50% of the tested population (LT50).71 While the As II and Sh1toxins were more efficient against T. ni larvae, with reductions in LT50 of 38.4% and 36%, respectively.71 In another example, the gene coding for the juvenile hormone esterase was integrated into the AcNPV genome. The hydrolysis of this hormone by esterase enzyme caused the triggering of a molt in the insect, as well as a stop in feeding. When recombinant viruses expressing this gene infected T. ni larvae, a reduction in feeding was observed, but no effect was detected on the LT50.72 Genes expressing toxins from other sources, such as B. thuringiensis 8-endotoxins, have also been integrated into the AcNPV genome.73 Genes expressing Cry protoxins from Bt aizawai and Bt. kurstaki were successfully expressed in recombinant viruses. Even, the typical Bt bipyramidal crystals were observed in the infected cell cultures. However, when recombinant viruses were tested against lepidopteran larvae, no difference was observed either in the LT50 or the LC50 (mean lethal concentration).74

It is important to mention that a gene from one baculovirus has been introduced into another baculovirus. This is the vef gene from the TnGV that was introduced into the AcNPV genome. The vef gene codes for the enhancin enzyme, which degrades the peritrophic membrane present in the insect midgut lumen thereby facilitating the contact of the virions with the midgut epithelial cells. When the recombinant virus was tested against T. ni larvae, a reduction of 22% in the LT50 was observed.75 The recombinant AcMNPV-enMP2 virus, expressing the MacoNPV enhancin gene under control of its native promoter was also developed and characterized.76 In T. ni larvae, the median lethal dose (LD50) of this recombinant virus was 4.4 times lower than that of AcMNPV wild-type virus. Conversely, a H. armigera single-nucleocapsid nucleopolyhe-drovirus (HaSNPV) was used to drive the expression of an insect-selective scorpion toxin (AaIT) at the egt gene locus of HaSNPV. Laboratory bioassays indicate that the median survival time (ST50) of H. armigera larvae was reduced 17-34% after infection with HaSNPV-AaIT in comparison to that of the wild-type virus.69 Finally, an AcMNPV recombinant was engineered to express the insect-selective toxin IT2 from the scorpion, Leiurus quinquestriatus. This recombinant virus elicited the response significantly faster than the common progenitor wild-type virus. The EC50 (effective concentration, EC) values of wild type AcMNPV, vAP10IT2 and vAPcmIT2 to 3rd-instar larvae of Trichoplusia ni were 1.00, 0.19 and 0.17 polyhedral inclusion bodies (PIBs) mm 3, respectively.77

Some tests using recombinant baculoviruses have been focused mainly on their prevalence in the field, rather than on their performance as bioinsecticides. This is due to some concerns related to the spread of the recombinant viruses in the environment and on the possibility of horizontal transmission of the transgene to other organisms. Still, very strict regulatory and safety requirements must be followed in many countries before the release of any genetically modified organisms, including viruses.

Recently, the potentially adverse effects of a recombinant AcMNPV (AcAaIT) were analyzed in rabbits and fishes. In the former, no marked changes in production of some enzymes were recorded. In addition, immuno-histochemical observation of tissues such as stomach, intestine, liver, kidney, brain, spleen and lung showed only slight changes. In the latter, no mortality was found in treated (AcAaIT) or untreated fish during the experimental period.78

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