Bioreactor Production Process of Spodoptera frugiperda multiple nucleopolyhedrovirus Biopesticide.

Spodoptera frugiperda (fall armyworm) is one of the most important maize pests in the world and the baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a natural pathogen of this pest, has been used as a biopesticide for its control. At present, in vivo strategies at the commercial scale are employed by multiplying the virus in the host insect in biofactory facilities; however, in vitro large-scale production is an interesting alternative to overcome the limitations of baculoviruses massal production. This study aimed to develop the process of the SfMNPV in vitro production by evaluating the effects of different multiplicities of infection (MOI) and nutritional supplements, morphological and molecular analysis of the infection on the growth of Sf9 cells and virus production. The Bioreactor Stirred Tank Reactor (STR) approach with glutamine-supplemented Sf-900 III serum free culture medium, combined with the MOI of 1.0, showed the best viral production performance, with a specific productivity above 300 occlusion bodies (OBs)/cell and volumetric productivity of 9.0 × 1011 OBs/L.

Optimization of Large-Scale Production of Chrysodeixis includens nucleopolyhedrovirus for Its Use as a Biopesticide.

The baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) is pathogenic to Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae) larvae, known as soybean looper, which is an important pest of soybean and bean. In this study, some parameters were tested to overcome the difficulties in the in vivo production of ChinNPV aiming to increase its use as a biopesticide. First, different combinations of larval instars (3rd and 4th instars), larval incubation temperatures (23 °C and 26 °C), and rearing densities (individually and 10 larvae/cup) were compared for larval weight and the production of occlusion bodies (OBs). A positive correlation (p< 0.001) was observed for OB production and larval weight. Fourth instar larvae produced more OBs than third instar larvae (p<0.05); however, no significant differences in OBs/larva (p>0.05) were observed for larvae kept in groups or individually. Therefore, a second assay was performed using fourth instar larvae incubated at 26 °C and two larval densities (10 larvae/cup and 40 larvae/cup). The losses of insects and OB production were evaluated as well as the influence of storage temperatures post-mortem (-20 °C, 4 °C, and 15 °C) in the OB yield. As expected, insect losses due to cannibalism or microbial contamination were greater (p<0.05) with the increase in larval density, although no difference was observed in OBs/larva (p>0.05). In addition, the storage temperature post-mortem did not influence the OB yield (p>0.05). The average production of ChinNPV OBs was 3×1010 OBs/40 larvae cup. The results demonstrate the viability of rearing C. includens in groups to enhance the mass production and reduce virus production costs.

Within-host interactions of Metarhizium rileyi strains and nucleopolyhedroviruses in Spodoptera frugiperda and Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Members of the family Baculoviridae have been quite successfully used as biocontrol agents against some lepidopterans. Likewise, a number of fungi are important natural enemies of these pests. An interesting approach to increase control efficacy could be the combination of a given nucleopolyhedrovirus (NPV) and a fungus, since they possess distinct modes of action. As a first step towards this goal, we assessed the interaction between NPV (either AgMNPV-79 or SfMNPV-6nd) and the entomopathogenic fungus Metarhizium rileyi (either CG1153 or CG381), using Anticarsia gemmatalis and Spodoptera frugiperda as hosts. In sequential applications of these pathogens, per os inoculation of an NPV (leaf discs with 2.5 × 104 occlusion bodies) either two days before or two days post-spraying of its counterpart fungal strain (5 × 103 conidia.cm-2 sprays) usually resulted in an antagonistic effect. When both pathogens were simultaneously applied at different combined dosages, usually an additive effect was seen. Interestingly, a number of dead larvae showing signs of co-infections (partially with soft integument and partially mummified) were recorded. However, mixes with lower dosages of both pathogens did not cause significantly higher insect mortalities compared to low dosages of the fungus applied alone. The advantages and disadvantages of the simultaneous applications of NPV and M. rileyi aiming at the management of either A. gemmatalis or S. frugiperda were discussed.

Characterization of a Chrysodeixis includens nucleopolyhedrovirus Isolate from Brazilian Cerrado and Assessment of its Co-Infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus

Abstract Chrysodeixis includens has become the major Lepidopteran pest of soybean crops, especially in the Brazilian Cerrado (savanna) region. A native isolate of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from this region, Buritis, MG, was assessed for its biological and molecular features. In addition, in vitro co-infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), another virus of an important soybean pest, was tested. The ChinNPV-Buritis isolate presented an average LC50 of 7,750 occlusion bodies (OBs)/ml of diet in C. includens larvae. Analysis of restriction endonuclease profiles of viral DNA revealed similarities with previously described ChinNPV isolates IE, IF, and IG from Brazil, although the presence of submolar bands indicates genetic heterogeneity. Optical microscopy analysis in conjunction with quantitative PCR (qPCR) demonstrated in vitro infection of this isolate in IPLB-SF-21AE, Sf9, and BTI-Tn-5B1-4 cell lines, but the amount of ChinNPV tends to decrease through serial passages. The qPCR method developed in this study successfully detected both AgMNPV and ChinNPV from cell culture and from infected larvae. The cell line Tn-5B1-4 is indicated for future development of in vitro production and co-infection studies.

Genome sequence of Erinnyis ello granulovirus (ErelGV), a natural cassava hornworm pesticide and the first sequenced sphingid-infecting betabaculovirus.

BACKGROUND: Cassava (Manihot esculenta) is the basic source for dietary energy of 500 million people in the world. In Brazil, Erinnyis ello ello (Lepidoptera: Sphingidae) is a major pest of cassava crops and a bottleneck for its production. In the 1980s, a naturally occurring baculovirus was isolated from E. ello larva and successfully applied as a bio-pesticide in the field. Here, we described the structure, the complete genome sequence, and the phylogenetic relationships of the first sphingid-infecting betabaculovirus. RESULTS: The baculovirus isolated from the cassava hornworm cadavers is a betabaculovirus designated Erinnyis ello granulovirus (ErelGV). The 102,759 bp long genome has a G + C content of 38.7%. We found 130 putative ORFs coding for polypeptides of at least 50 amino acid residues. Only eight genes were found to be unique. ErelGV is closely related to ChocGV and PiraGV isolates. We did not find typical homologous regions and cathepsin and chitinase homologous genes are lacked. The presence of he65 and p43 homologous genes suggests horizontal gene transfer from Alphabaculovirus. Moreover, we found a nucleotide metabolism-related gene and two genes that could be acquired probably from Densovirus. CONCLUSIONS: The ErelGV represents a new virus species from the genus Betabaculovirus and is the closest relative of ChocGV. It contains a dUTPase-like, a he65-like, p43-like genes, which are also found in several other alpha- and betabaculovirus genomes, and two Densovirus-related genes. Importantly, recombination events between insect viruses from unrelated families and genera might drive baculovirus genomic evolution.

Detection and identification of baculovirus pesticides by multitemperature single-strand conformational polymorphism.

The method of single-strand conformational polymorphism (SSCP) was modified in our laboratories for the characterization of baculoviruses, insect viruses with great potential for use as bioinsecticides in biological protection programs. A series of primers were synthesized after the comparison of the polyhedrin gene sequences of over 20 baculoviruses. Polyhedrin is a highly conserved protein which is responsible for the persistence of the virus in the environment. Universal primers were designed which could be used in polymerase chain reactions (PCR) containing genomic DNA from an array of nucleopolyhedrosis viruses (NPVs) including these which are used as biopesticides against important pests of forests and crops, such as Anticarsia gemmatalis, Spodoptera frugiperda, Lymantria dispar, Lymantria monacha and many others. PCR products were denatured and subjected to single-strand DNA electrophoresis at variable temperatures (MSSCP) where, after silver staining, they gave ssDNA band patterns characteristic for each baculovirus species. This technique can be potentially applied to detect baculoviruses in insects collected in the field, as well as to plant tissues and the excrements or bodies of predators without need for sequencing the PCR products. Sometimes MSSCP can be used not only for species determination but also as an indication of genomic variability which can be related to infectivity.