Recombinant Helicoverpa armigera nucleopolyhedrovirus with arthropod‐specific neurotoxin gene RjAa17f from Rhopalurus junceus enhances the virulence against the host larvae

A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV) expressing the insect‐selective neurotoxin (RjAa17f) from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP‐glucosyltransferase gene (egt) using λ‐red homologous recombination system. Another egt deleted control HearNPV was constructed in a similar way by inserting egfp gene into the egt locus. One‐step viral growth curve and viral DNA replication curve analysis confirmed that the recombination did not affect the viral growth and DNA replication in host cells. There is no discernable difference in occlusion‐body morphogenesis between RjAa17f‐HearNPV, Egfp‐HearNPV and HZ8‐HearNPV, which was confirmed by transmission electron microscopy analysis. However, the insecticidal activity of RjAa17f‐HearNPV is enhanced against the third instar H. armigera larvae according to the bioassay on virulence comparison. There is a dramatic reduction (56.9%) in median lethal dose (LD₅₀) and also a reduction (13.4%) in median survival time (ST₅₀) for the recombinant RjAa17f‐HearNPV compared to the HZ8‐HearNPV, but only a 27.5% reduction in LD₅₀ and 10.1% reduction in ST₅₀ value when Egfp‐HearNPV is compared with HZ8‐HearNPV. The daily diet consumption analysis showed that the RjAa17f‐HearNPV was able to inhibit the infected larvae feeding compared with the egt minus HearNPV. These results demonstrated that this novel recombinant RjAa17f‐HearNPV could improve the insecticidal effect against its host insects and RjAa17f could be a considerable candidate for other recombinant baculovirus constructions.     

Functional analysis of the Autographa californica nucleopolyhedrovirus IAP1 and IAP2

The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in de-tail. AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4 (Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV (HearNPV) infection and rescued the replication of HearNPV and BV production in these cells. Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection. Recombinant HearNPVs ex-pressing AcMNPV iap1, iap2 and p35, respectively, not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells. However, the iap1, iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production. These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.     

Identification of the epitopes of monoclonal antibodies against P74 of Helicoverpa armigera nucleopolyhedrovirus

P74 is a per os infectivity factor of baculovirus. Here, we report the production of three monoclonal antibodies （mAbs）, denoted as 20D9, 20F9 and 21E1, raised against P74 of Helicoverpa armigera nucleopolyhedrovirus （HearNPV）, and the identification of their recognition epitopes. The full-length P74, without the transmembrane domains at the C-terminus, was first divided into three segments （N, M and C, respectively）, based on the proposed cleavage model for the protein, which were then expressed individually. Western blot analyses revealed specific cross-reactions with the N fragment, for both 20D9 and 21E1. Extensive truncation, followed by prokaryotic expression, of the P74 N fragment was then performed in order to screen for linear epitopes of P74. The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219, respectively. In addition, immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells. These findings will facilitate further investigations of the proteolytic processing of HearNPV P74, and of its involvement in virus-host interactions.     

Evaluation of Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) for Control of Helicoverpa armigera (Lepidoptera: Noctuidae) on Citrus in South Africa

A concentration of 1.15×10⁷ OBs/mL of HearNPV, sprayed using a knapsack, resulted in a 100% reduction in Helicoverpa armigera larval infestation within 7 days on tomato plants in a hot house environment. A 10-fold lower concentration, 1.15×10⁶ OBs/mL, resulted in a 100% reduction within 16 days. These two concentrations reduced damage to tomatoes by 81 and 69%, respectively. In two field trials on navel oranges, the lower concentration and an additional even lower concentration of 7.26×10⁵ OBs/mL, both resulted in a 100% reduction in H. armigera infestation within 14 days or longer. H. armigera damage to fruit was reduced by up to 84 and 75%, respectively, in the two trials. Rejection for export was reduced by 96 and 62%. This could imply a saving of US$339 per hectare compared to the untreated control. These results were better than those achieved with Bacillus thuringiensis and various standard chemical insecticides used in the same trials. Reasons for these results are discussed.