Stability of a Spodoptera frugiperda nucleopolyhedrovirus deletion recombinant during serial passage in insects

Abstract

The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.

FIG. 1.

Mean concentrations of DNA (ng/ml) extracted from samples of 5 × 10⁸ OBs of SfWT, Sfbac, and Sf29null bacmids at each passage. Three different DNA extractions per virus were performed at each passage. DNA concentrations of each sample were measured three times. Different letters above the bars indicate significant differences between treatments and passages (Mann-Whitney test, P ≤ 0.05).

FIG. 2.

ODV contents in 5 × 10⁸ OBs of SfMNPV wild-type (SfWT), SfMNPV bacmid (Sfbac), and Sf29null bacmid viruses at each passage (P0, P1, P2, P3, P4, and P5). Sf21 cells were serially infected (1:5, 1:25, 1:125, and 1:625) with ODVs released from OBs. ODV titers (ODV/ml) were calculated by end point dilution in triplicate. Different letters above the bars indicate significant differences between treatments and passages (Mann-Whitney test, P ≤ 0.05).

FIG. 3.

Semiquantitative PCR analysis of the relative proportions of kanamycin resistance cassette- and Sf29-carrying genomes in the OBs from the SfMNPV wild-type (SfWT), SfMNPV bacmid (Sfbac), and Sf29null bacmid viruses at each passage (P0, P1, P2, P3, P4, and P5). Percentages next to amplicons indicate the relative proportions of each product as estimated by densitometric analysis (Scion Image program). M, molecular mass marker.

FIG. 4.

Numbers of colonies grown in chloramphenicol (Cm) and chloramphenicol-kanamycin (Cm + Kan) plates, after transformation of DH5B10 electrocompetent cells with 150 ng of Sfbac (A) and Sf29null (B) DNAs at each passage (P0, P1, P2, P3, P4, and P5) in triplicate. Three DNA extractions were performed per virus at each passage. The proportions of colonies grown in chloramphenicol relative to colonies grown in chloramphenicol-kanamycin after transforming 150 ng of Sf29null DNA were 1:1, 1:0.71, 1:0.31, 1:0.17, 1:0.09, and 1:0.05 during passages P0 to P5, respectively.