| Bacground:Mosquito-borne diseases such as malaria, dengue, and yellow fever constitute a major public health problem, because they are responsible for a considerable amount of morbidity and mortality. Vector control is a key means of combating mosquito-borne diseases. However, the traditional method of the application of chemical pesticides has generated complex problems because of the high level of toxicity caused to the environment, consequent safety risks for humans, and insecticide resistance in mosquitoes. Growing concern over the environmental effects of pesticides has encouraged the development of valuable alternative approaches to control these diseases. Further research into the biology of the pathogens that infect mosquitoes may offer potential strategies. They could be used as biological control agents or foreign gene carriers that act either to reduce mosquito populations or the vectorial capacity of mosquitoes.Mosquito densoviruses (MDVs) are small, autonomous, non-enveloped DNA viruses with small single-stranded DNA genomes, and the majority of them belong to the Brevidensovirus genus in the subfamily Densovirinae within the Parvoviridae family. Since the first MDV. Aedes aegypti densovirus (AeDNV) was discovered at Kiev National University in1972, a number of MDVs have been isolated from persistently infected mosquito cell lines, laboratory colonies, or natural populations. These viruses are often pathogenic to their hosts; they replicate in the nuclei of cells of mosquitoes and cause characteristic histopathological signs, which include hypertrophied, densely stained nuclei. Most importantly, MDVs have a high specificity for mosquitoes as a host, are relatively stable in the environment, and have the potential to spread and persist in mosquito populations. All of these features make them attractive agents that could be developed as bioinsecticides for the control of mosquitoes. However, like most wild-type (wt) microbial insecticides, there are several limitations for the use of wt MDVs as insecticides that have restricted their commercial use. Their major disadvantage is poor efficacy. They are slow to act and their killing efficiency operates in a dose-dependent manner, which means that a significant insecticide activity can only be achieved when the virus titer exceeds their native level. To solve this problem, insect viruses are often genetically modified to express insecticidal genes. As a result, the efficiency of recombinant viruses was enhanced and is able to incapacitate its host in a shorter period of time.Scorpion insect-specific toxin is strictly selective with a high insect toxicity, which means that they can be considered as an ideal candidate for pest control. It was reported previously that the insecticidal properties of Autographa california nuclear polyhedrosis virus (AcNPV) and Periplaneta fuliginosa densovirus (PfDNV) were enhanced effectively by introducing scorpion insect-specific toxin. An excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) is an insect-selective single-chain neurotoxic polypeptide that is composed of88amino acid residues cross-linked by four disulfide bridges. It has been shown to affect insect neuronal sodium conductance by binding to excitable sodium channels.Objective:In this report, we constructed a series of recombinant AeDNV that expressed BmK IT1. A green fluorescent protein (GFP) gene was also ligated to the C-terminus of the BmK IT1gene as a screening marker. Bioassays were carried out in vitro to measure the insecticidal effect of recombinant viruses on Ae. albopictus larvae.Methords:The BmK IT1gene, which includes a54-bp sequence that encodes the signal, was redesigned based on the codon usage frequency preferred by Ae. albopictus. The different BmK IT1expression vector p7NSl-BIT-GFP, p61NTS-BIT-GFP and p7NS1-BITA11were constructed, and expression of BmK IT1in C6/36cell was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblots. Expression and intracellular localization of BmK IT1-GFP fusion proteins were dectected by DAPI and GFP under an inverted fluorescence microscope. Recombinant viruses (vAep7NS1-BIT-GFP, vAep61NTS-BIT-GFP, and vAep7NS1-BIT) were generated along with wt AeDNV by cotransfecting the infectious clones, p61NTS-BIT-GFP, p7NSl-BIT-GFP, and p7NSl-BIT, individually with helper plasmid pUCA into C6/36cells. Every batch of mosquitoes was examined by conventional PCR to ensure that the experimental mosquitoes were free from MDVs (data not shown). First-instar Ae. albopictus larvae were exposed by introducing them into the beaker that contained the virus mix stocks. One-step RT-PCR was performed on the total RNA using BmK IT1gene-specific primers. The rabbit polyclonal IgG anti-BmK IT1was used as the primary antibody to detect the expression of BmK IT1protein. The copy number of recombinant virus and AeDNV in three kinds of mix stocks were confirmed by real-time PCR, and then the copy number of recombinant virus and AeDNV in three kinds of mix stocks were adjusted to the same ratio by applying different volumes of pure AeDNV. First-instar larvae were randomly divided into recombinant virus. The median lethal concentration (LC50) of first-instar mosquito larvae was determined after being exposed to wt and three kinds of recombinant virus at a series of concentrations that ranged from1×107to1×1011copies/mL. The median survival time (LT50) was calculated from the time-mortality curve of larvae that were infected by recombinant virus and wt virus at the concentrations of1.0×1010and1.0×1011copies/mL, respectively. The sublethal effect was determined by the variances in time of pupation and emergence, the rate of pupation, and their emergence at the concentrations of1.0×108copies/mL. Each experiment was repeated three times.Results:We identified the expression of BmK IT1by RT-PCR and immunoblotting with the use of gene-specific primers and anti-BmK IT1polyclonal immunoglobulin G (IgG), respectively. The RT-PCR revealed that BmK IT1mRNA was detected in cell line and mosquito body that were transfected or transduced with BmK IT1constructs or rrecombinant virus. Western blot analysis identified that the anti-BmK IT1polyclonal IgG reacts specifically band, respectively. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT50and LD50bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.Conclusion:In summary, we have showed that genetic engineering is an effective way to increase the virulence of wt AeDNV, and the recombinant virus may be valuable as a vector control agent in the future. However, whether this system will become a practical method for insect control remains to be seen. The main concern in the use of recombinant virus with toxin genes is safety, including the issue of toxin specificity and the risk of gene flow into non-target organisms through unintended infection. Even though MDV is highly specific for mosquitoes as hosts, the high selectivity of the toxin to insects together with the uniqueness of transcriptional activation of MDV promoters in permissive insect cells provides safety at multiple levels. Further data to support the environmental safety of genetically modified AeDNV are still required. Analogous research of insect viruses, including the genetic modification of baculovirus to express scorpion toxin, has yet to be translated into practice. This is largely a result of commercial and political issues rather than technical limitations. Although MDV has a number of attractive attributes as a microbial control agent, there are a number of questions that remain to be answered before its maximal value can be realized. |
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