Abstract
One of the interesting features of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genome is the absence of chitinase (chiA) and cathepsin (v-cath) genes. This characteristic may be responsible for the lack of liquefaction and melanization in A. gemmatalis larvae killed by AgMNPV-2D infection. This study aimed to test the hypothesis that CHIA and V-CATH proteins from Choristonera fumiferana DEF multiple nucleopolyhedrovirus (CfDEFNPV) are able to liquefy and melanize the cuticle of A. gemmatalis larvae infected by a recombinant AgMNPV containing chiA and v-cath genes inserted in its genome. A fragment from the CfDefNPV genome containing chiA and v-cath genes was inserted into the genome of AgMNPV-2D. The recombinant virus (vAgp2100Cf.chiA/v-cath) was purified and used to infect insect cells and larvae. Transcripts of v-cath and chiA genes were detected along the infection of insect cells by qRT-PCR, from early to late phases of infection. The analysis of A. gemmatalis larvae killed by vAgp2100Cf.chiA/v-cath infection confirmed the hypothesis proposed. The vAgp2100Cf.chiA/v-cath showed higher insecticidal activity against third instar A. gemmatalis larvae when compared to AgMNPV-2D. The mean time to death was also lower for the vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D at 10 days post infection. Occlusion body production was higher in A. gemmatalis larvae infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. Enzyme assays showed higher chitinase and cysteine protease activities in insect cells and insects infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. The introduction of chiA and v-cath genes into the genome of AgMNPV improves its insecticidal activity against A. gemmatalis larvae and this recombinant virus could be used as an alternative to the wild type virus to control this important insect pest.
Highlights
Brazil is considered the second largest soybean producer and the third largest exporter of agricultural products in the world [1]
Considering that most Alphabaculoviruses have in their genomes the chiA and v-cath genes, and given the importance of the action of these proteins in the virus-host interaction resulting the insects final liquefaction and further spread of viral progeny, this study proposed to test the hypothesis that the expression of proteins CHIA and V-CATH from CfDefNPV in A. gemmatalis larvae during infection by a recombinant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) induces liquefaction of the insect tegument and its melanization
Construction of AgMNPV recombinant virus containing the chiA and v-cath genes p2100Cf.chiA/v-cath plasmid DNA was cotransfected with DNA from the recombinant baculovirus vAgGalA2 in insect cells (UFL-AG-286)
Summary
Brazil is considered the second largest soybean producer and the third largest exporter of agricultural products in the world [1]. An alternative to the use of chemical pesticides in the crops to control insect pests is the use of biological agents, such as the baculoviruses [2]. These viruses contain rod-shaped virions and a large, circular, super coiled double-stranded DNA genome ranging from 80 to 200 kilobases (kb) [3]. A peculiarity of baculoviruses is the production of two phenotypically distinct viruses in a single cycle of infection: the budded viruses (BVs), which are responsible for the systemic infection within the host, from cell to cell; and the occlusionderived viruses (ODVs), which are occluded in a proteinaceous occlusion body (OB), known as polyhedra, responsible for horizontal transmission from insect to insect [6]. The disintegration of the host is facilitated by the synergistic interaction between the viral proteins V-CATH (a cysteine protease) and CHIA (a viral chitinase) which are codified by two genes present in several baculovirus genomes [9,10]
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