Recent advances in biological control of pest insects by using viruses in China

Insect viruses are attractive as biological control agents and could be a feasible alternative to chemical insecticides in the management of insect infestations. This review describes recent advances in the development of wild-type and genetically modified viruses as insecticides. A new strategy of application of insect viruses in China is reviewed. Also, the assessment of biosafety of genetically modified Helicoverpa armigera Nucleopolyhedovirus (HearNPV) is emphasized as a case-study. Foundation itims: The 863 projects (2006AA10A210) from MOST.     

重组病毒杀虫剂应用研究进展

应用分子生物学技术可以将昆虫特异性的毒素基因、某些酶基因等外源基因插入昆虫病毒基因组,或通过改造昆虫病毒基因组等方法构建重组病毒杀虫剂,提高杀虫效果。温室及田间释放实验证实,重组病毒杀虫剂可以显著提高现场防治效果。连续多代抗性筛选实验表明,宿主被诱导产生对重组病毒杀虫剂抗性的速度低于野生型病毒杀虫剂。采用在剂型中添加光增白剂等保护剂、在基因组中插入具有增效作用的基因、应用病毒增强蛋白等技术可以提高重组病毒杀虫效果。随着基因工程技术的发展和安全性研究的深入,以重组杆状病毒为主的重组昆虫病毒杀虫剂的应用研究正面临着突破。     

Not all GMOs are crop plants: non-plant GMO applications in agriculture

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.     

Persistence of viruses and DNA in soil

With growing environmental concerns over the use of chemical pesticides for insect control in both agriculture and forestry, increased emphasis is being placed on the development of alternative, biological pesticides such as genetically modified baculoviruses. Before the large-scale use of genetically modified viruses (GMV) can be realized, fate of GMV and their DNA in soil should be investigated. There are a number of factors that have the potential to affect persistence of both wild-type and genetically modified viruses and their DNA in soil. In this mini-review, the persistence of viral particles and DNA in soil is examined with particular emphasis on baculoviruses.     

Persistence of viruses and DNA in soil

With growing environmental concerns over the use of chemical pesticides for insect control in both agriculture and forestry, increased emphasis is being placed on the development of alternative, biological pesticides such as genetically modified baculoviruses. Before the large-scale use of genetically modified viruses (GMV) can be realized, fate of GMV and their DNA in soil should be investigated. There are a number of factors that have the potential to affect persistence of both wild-type and genetically modified viruses and their DNA in soil. In this mini-review, the persistence of viral particles and DNA in soil is examined with particular emphasis on baculoviruses.     

Behavior of a recombinant baculovirus in lepidopteran hosts with different susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.     

New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus

Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner.     

Can microbial-based insecticides replace chemical pesticides in agricultural production?

Extensive use of chemical insecticides to control insect pests in agriculture has improved yields and production of high-quality food products. However, chemical insecticides have been shown to be harmful also to beneficial insects and many other organisms like vertebrates. Thus, there is a need to replace those chemical insecticides by other control methods in order to protect the environment. Insect pest pathogens, like bacteria, viruses or fungi, are interesting alternatives for production of microbial-based insecticides to replace the use of chemical products in agriculture. Organic farming, which does not use chemical pesticides for pest control, relies on integrated pest management techniques and in the use of microbial-based insecticides for pest control. Microbial-based insecticides require precise formulation and extensive monitoring of insect pests, since they are highly specific for certain insect pests and in general are more effective for larval young instars. Here, we analyse the possibility of using microbial-based insecticides to replace chemical pesticides in agricultural production.     

昆虫碱性磷酸酶的研究进展

碱性磷酸酶存在于昆虫的头、唾液腺（唾液）、肠道、马氏管、表皮、血淋巴、脂肪体、生殖系统、附肢等部位,广泛参与了昆虫的发育、神经传导、激素合成、物质代谢、滞育、社会型昆虫亚种形成等过程。同时碱性磷酸酶与昆虫抗性有关,特别涉及到对Bt制剂的阻滞作用,其本身也是某些农药的靶标酶,某些生物源化合物及病毒、真菌也可以影响其活性。昆虫碱性磷酸酶的研究,将有助于提高对昆虫生化机制及代谢过程的认识,并为害虫治理和资源昆虫饲养提供新的思路。本文综述了国内外对昆虫碱性磷酸酶的研究状况,并描述了昆虫碱性磷酸酶的生化性质及其与生理功能的关系。     

Evolution of resistance to penicillin and cephalosporin antibiotics

Microorganisms belong to the main regulators of insect populations under natural conditions. They perform this function in several ways. In the first place, bacterial metabolites exist which are as potent as chemical insecticides. In the second, entomopathogenic fungi and viruses spread deadly infections among insect populations. The use of advanced techniques in molecular biology, genetic engineering and biotechnology is increasingly expanding the potential of microorganisms as agents of insect pest control.     

Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in Trichoplusia ni Indicates High Multiplicity of Cellular Infection

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.     

Can synthetic pesticides be replaced with biologically-based alternatives?—an industry perspective

Agricultural chemical companies have invested in the discovery and development of biological pesticides to complement synthetic pesticides for the control of insects, diseases, and weeds on agronomic and horticultural crops. For plant disease control, companies envisage biological fungicides entering markets where they have the best chance of performing and which are most receptive to using biological control methods. Fewer regulatory requirements can mean faster registration for a biological than a synthetic pesticide. However, industry’s requirements for competitive performance, effective formulations, and economic production can mean significant investments in time and money for a biological pesticide, although total investment may be less than for a synthetic pesticide. One biocontrol project in which industry has invested is baculoviruses for insect control. Insect baculoviruses, genetically modified to kill insects faster than wild-type viruses, are attractive biocontrol agents because their selectivity to insect pests and safety to beneficial insects and mammals enable them to compete with synthetic insecticides. Industry is looking for similar biocontrol opportunities in disease control. Biocontrol agents for seedling disease, root rot, and postharvest disease control have been registered by the EPA and are trying to compete with synthetic fungicides for market share. To date, effective biocontrol agents have not been identified for the control of serious foliar diseases, such as grape downy mildew, potato late blight, wheat powdery mildew, and apple scab. Farmers must rely on synthetic fungicides and agronomic methods to control these diseases for the foreseeable future. Received 06 February 1997/ Accepted in revised form 01 June 1997     

Genetically modified microbial symbionts as arthropod pest controllers: risk assessment through the European legislations

The genetic modification and applied use of microbial symbionts have been identified as novel tools to protect beneficial insects such as pollinators or parasitoids or to fight insects that constitute pests or are vectors of infectious diseases. The deliberate release of insect pest or disease vector control products containing genetically modified micro‐organisms (GMMs) can raise questions about health and environmental safety. Different national and international authorities have established legal requirements to ensure the safe use of conventional pesticides and insecticides as well as GMMs. A key requirement is to conduct a scientific risk assessment to determine whether the product is safe to be placed in the market. In this study, we address the legal framework, the regulatory requirements, and the criteria for the environmental risk assessment of GM symbionts that currently apply within the European Union.     

Insect control with baculoviruses

Baculoviruses have been researched extensively for insect control. Three of their features have been particularly attractive: their host specificity and consequential environmental safety, their virulence in host insects, and their capability for causing disease epizootics. There have been four approaches to their use for insect control: as microbial insecticides for short-term insect population suppression, through seasonal colonization or a recurring "booster shot" for control of more than one pest generation, through introduction-establishment where the viral species or strain was not indigenous, and through environmental manipulation to make the ecosystem more favorable for viral epizootics. Actual usage of baculoviruses in pest management has been disappointing, particularly with the microbial insecticide approach, primarily for three reasons: economics, slow speed of kill, and adverse effects of the environment on the viruses. The recombinant-DNA revolution has greatly increased the prospects for baculoviruses in insect pest management.     

鳞翅目昆虫抗病毒免疫反应的研究进展

鳞翅目昆虫种类繁多,对农业生产和人类生活产生重大影响,宿主昆虫与病毒相互关系的研究对于利用病毒杀虫剂进行害虫治理和益虫病毒性疾病的预防具有重要意义.因此,鳞翅目昆虫与病毒的互作研究显得尤为重要,宿主昆虫的免疫系统在抗病毒感染过程中发挥着关键作用,对病毒产生不同程度的免疫反应.本文综述了昆虫围食膜和中肠对病毒入侵的防御作用,病毒进入体腔后昆虫所产生的细胞免疫和体液免疫反应,以及RNAi、细胞的自噬与凋亡、Toll、Imd、JAK-STAT和STING信号通路等相关的抗病毒免疫途径,并对昆虫抗病毒免疫研究的制约因素和未来鳞翅目昆虫抗病毒免疫的研究重点进行了讨论,以期为害虫的生物防治和益虫疾病的防控提供理论依据.     

Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.     

Transgenesis and paratransgenesis to control insect-borne diseases: Current status and future challenges

Insect-borne diseases cause significant human morbidity and mortality. Current control and preventive methods against vector-borne diseases rely mainly on insecticides. The emergence of insecticide resistance in many disease vectors highlights the necessity to develop new strategies to control these insects. Vector transgenesis and paratransgenesis are novel strategies that aim at reducing insect vectorial capacity, or seek to eliminate transmission of pathogens such as Plasmodium sp., Trypanosoma sp., and Dengue virus currently being developed. Vector transgenesis relies on direct genetic manipulation of disease vectors making them incapable of functioning as vectors of a given pathogen. Paratransgenesis focuses on utilizing genetically modified insect symbionts to express molecules within the vector that are deleterious to pathogens they transmit. Despite the many successes achieved in developing such techniques in the last several years, many significant barriers remain and need to be overcome prior to any of these approaches become a reality. Here, we highlight the current status of these strategies, pointing out advantages and constraints, and also explore issues that need to be resolved before the establishment of transgenesis and paratransgenesis as tools to prevent vector-borne diseases.     

Genomics spawns novel approaches to mosquito control

In spite of advances in medicine and public health, malaria and other mosquito-borne diseases are on the rise worldwide. Although vaccines, genetically modified mosquitoes and safer insecticides are under development, herein we examine a promising new approach to malaria control through better repellents. Current repellents, usually based on DEET, inhibit host finding by impeding insect olfaction, but have significant drawbacks. We discuss how comparative genomics, using data from the Anopheles genome project, allows the rapid identification of members of three protein classes critical to insect olfaction: odorant-binding proteins, G-protein-coupled receptors, and odorant-degrading enzymes. A rational design approach similar to that used by the pharmaceutical industry for drug development can then be applied to the development of products that interfere with mosquito olfaction. Such products have the potential to provide more complete, safer and longer lasting protection than conventional repellents, preventing disease transmission by interrupting the parasite life cycle.