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Abstract: Light and stereomicroscopy examinations of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV)-infected insects were performed in order to follow infection in its host, A. gemmatalis. Fourth-instar A. gemmatalis larvae were infected by administration of occluded virus (polyhedra) from two recombinant AgMNPV viruses (vAgEGTΔ-lacZ or vAgGalA2) directly into the larvae foregut. The recombinant virus vAgEGTΔ-lacZ has the β-galactosidase gene (lac-Z) of Escherichia coli under the control of a constitutive promoter (hsp70 from Drosophila melanogaster). The vAgGalA2 virus has the reporter gene lac-Z under the control of the AgMNPV very late polyhedrin gene promoter. At different times post-infection (p.i.) the infected larvae were dissected, fixed, and the product of the expression of the lac-Z gene detected by incubating the insects in a buffer containing X-gal. This allowed us to follow the infection through the blue cells (due to the degradation of X-gal by the enzyme Lac-Z). Insect larvae inoculated with polyhedra from the recombinant viruses showed midgut cells to be infected first, followed by tracheal cells, hemolymph, fat body, Malpighian tubules and brain cells. The infection was similar for the two recombinant viruses, with blue cells appearing earlier in insects infected with the vAgEGTΔ-lacZ virus when compared to the vAgGalA2 virus. [Copyright &y& Elsevier]

Highlights: [•] We analyze the genetic stability of per os infection factors (pif) genes from AgMNPV isolates. [•] The pif genes have few genetic differences and the pif-2 gene shows the most variation. [•] The pif genes sequences analyzed were conserved along 20years of virus application in the field. [Copyright &y& Elsevier]

Abstract: Malpighian tubules constitute the main excretion organ of insects. Infection by egt− recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt− , and used another two recombinants (one egt− ) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt− viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host. [Copyright &y& Elsevier]

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×108 OBs/g/larva and 40.7×108 OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC50) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV. [Copyright &y& Elsevier]