Progress 09/01/09 to 08/31/15
Outputs
Target Audience:Researchers with interests in insect physiology, insect virology and insect pest management. Changes/Problems:No additional research was conducted on objective2 during the course of the current project funding period. A decision was made to focus on objective 3 instead as significant advances were made for that objective. What opportunities for training and professional development has the project provided?Jimena Carrillo-Tripp, a postdoctoral research associate, and Diveena Vijayendran, a graduate student, have both received training during the course of this project. Collaborators: W. Allen Miller, Iowa State University; David Murhammer, University of Iowa, IA. How have the results been disseminated to communities of interest?Research results from this project were presented by participants and discussed at the 2013 Keystone meeting on RNA Interference, the All Iowa Virology Symposium, the annual meeting of the American Society for Virology and the annual meeting of the International Society for Invertebrate Pathology. Data have also been published in peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall objective of this project is to investigate the potential for exploitation of viruses for development of novel strategies for insect pest management. A significant portion of U.S. agriculture is lost to insect pests on an annual basis, and new, environmentally safe methods are needed to suppress damaging insect populations. During the five year duration of this project, significant advances have been made in 1) the understanding of virus-insect interactions that will facilitate the use of insect viruses for pest management purposes. Increased knowledge has been gained of factors that determine which pest insects are infected by baculoviruses, which can be used to control lepidopteran (moth) pests; 2) Several new viruses of aphids have been identified and characterized. Knowledge of aphid viruses, their characteristics and distribution between different insect orders has increased. This is important because aphids are pervasive in temperate agriculture and aphid-related crop damage results from direct feeding by the aphid, and aphid transmission of plant viruses results in considerable economic loss. Objective 1: determine the molecular bases for baculovirus host range within the Lepidoptera (moths) To assess the role of baculovirus occlusion-derived envelope protein, ODV-E56 in host range, the ODV-E56 gene of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) was substituted with the ODV-E56 gene of the virus, Rachiplusia ou nucleopolyhedrovirus (RoNPV), a closely related virus that is significantly more virulent towards some host species than AcMNPV. The role of ODV-E56 was evaluated and found to be essential for oral infectivity to larvae of the moth pest, Heliothis virescens. Although essential for oral infectivity, the ODV-E56 protein itself is not required for binding or fusion of the virus to the insect gut epithelial cells. Replacement of the AcMNPV odv-e56 gene with the RoMNPV orthologue did not increase virulence against these two species. Understanding the interaction of a baculovirus with the insect gut may in the long term allow for genetic manipulation of baculovirus host range. This would be a particularly useful tool for management of multiple pests that are not all equally susceptible to a baculovirus. An additional outcome of this project for the potential management of moth pests was the accidental discovery of a virus in a cell line. During the course of this research, a virus was accidentally discovered persistently infecting a laboratory cell line. This virus was identified and characterized. We identified and characterized the iflavirus, Lymantria dispar iflavirus 1 (LdIV1) that persistently infects an insect cell line. This virus may have potential for use in suppression of damaging gypsy moth populations. LdIV1 may provide for a novel approach for suppression of gypsy moth populations which feed on hardwood trees and severely impact forests in north America Objective 2: investigate the potential of basement membrane-degrading proteases to enhance baculovirus insecticides and for use in pest management No additional research was conducted on this objective during the course of the current project funding period. A decision was made to focus on objective 3 instead as significant advances were made for that objective. Objective 3: assess the potential use of viruses for management of aphid pests Aphid viruses have potential for use in the management of pestiferous aphids, which cause considerable economic loss by reducing crop yields in temperate climates. Identification of new viruses of aphids and characterization of the virulence of all aphid viruses will facilitate identification of aphid viruses that can be used for aphid management. During the course of this project significant advances were made for two novel aphid viruses in particular: Aphid lethal paralysis virus AP1 (ALPV-AP1), and Aphis glycines virus 1 (AGV1). Small RNA sequences from multiple aphid species were sequenced and analyzed. EST data were examined for the presence of virus sequences. Numerous insect cell lines were screened for their ability to support replication of the aphid viruses identified, and aphid populations were screened for the presence of the viruses identified. Virus sequences were identified from small RNA sequence data, along with unique, aphid-specific miRNA that may have potential for future aphid management. Analysis of sRNA reads, and EST sequences resulted in identification of two Aphid lethal paralysis virus (ALPV)-like viruses (ALPV-AP1, and ALPV-AP2) from the pea aphid (Acyrthosiphon pisum), a Myzus persicae virus-like virus from the green peach aphid, and an ALPV-like virus from western corn rootworm, Diabrotica virgifera virgifera. These results indicate that ALPV-like viruses are widespread across multiple insect orders. This distribution across multiple pestiferous species is attractive for pest management purposes. ALPV-AP1: ALPV-AP1 is prevalent in pea aphid populations. On screening of multiple insect cell lines, a 60-fold increase in virus titer three days after addition of ALPV-AP1 RNA to the DU182A cell line was detected indicating that this cell line can support replication of ALPV-AP1. Identification of a cell line that supports replication of ALPV-AP1 may facilitate production of this virus for use in suppression of damaging aphid populations. AGV1: The soybean aphid, Aphis glycines has caused significant economic loss in the Midwest over the course of the past decade. We identified several viruses that infect this species, including the newly described Aphis glycines virus 1 (AGV1). The genome sequence of AGV1 was determined, along with biological characteristics including a 100% vertical transmission rate. AGV is widely distributed across multiple states including Iowa, Ohio and Michigan. The next step will be to determine how virulent these viruses are against aphids to assess whether any can be used for management of pestiferous aphids.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Researchers with interests in insect physiology, insect virology and insect pest management. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Jimena Carrillo-Tripp, a postdoctoral research associate, and Diveena Vijayendran, a graduate student have both received training during the course of this project. How have the results been disseminated to communities of interest? Two research manuscripts have been published in peer reviewed journals, describing this research. What do you plan to do during the next reporting period to accomplish the goals? Research on AGV and a second manuscript on LyDiV are planned for completion of this work.
Impacts
What was accomplished under these goals?
Impact: Aphid viruses have potential for use in management of pestiferous aphids, which cause considerable economic loss by reducing crop yields in temperate climates. The virus LyDiV may also provide for a novel approach for suppression of gypsy moth populations which deleteriously impact forests. During this reporting period, research was continued on two aphid viruses and on a newly discovered gypsy moth virus. The full length Aphis glycines virus (AGV) genome sequence is now known with the 5' and 3' end sequences determined by using Rapid Amplification of cDNA ends (RACE). AGV was not detected in soybean plants infested by soybean aphids, suggesting that AGV is not horizontally transmitted via the plant, in contrast to several other viruses that infect aphids. AGV replication is regulated by the soybean aphid RNA interference (RNAi) pathway as indicated by detection of AGV-derived small interfering RNA (siRNA). The presence of Aphid lethal paralysis virus (ALPV) in aphids infected with AGV resulted in an increase in AGV-derived siRNA relative to aphids infected with AGV alone, suggesting an indirect impact of ALPV on AGV infection via the RNAi pathway. The novel virus Lymantria dispar iflavirus 1 (LdIV1) isolated from a gypsy moth cell line (IPLB-Ld652Y) was characterized and data published. LdIV1 was discovered infecting the cell line and individual gypsy moths without inducing overt signs of infection. Nonetheless, virions isolated from the cell culture were infectious in different gypsy moth virus-free cell lines (IPLB-Ld65 and IPLB-LdFB). The infection of IPLB-Ld652Y was switched from covert to acute when a viral suppressor of RNAi was added to the cells. These results show the potential for use of LdIV1 as a biocontrol agent of the gypsy moth by 1, the de novo infection of virus-free insects and 2, by the induction of acute infection in individuals covertly infected with the virus. Analyses of small RNA populations in the covertly infected cell line (IPLB-Ld652Y) compared to one acutely infected cell line (IPLB-Ld652) are under way to better understand the molecular mechanisms of LdIV1 infection. The official inclusion of LdIV1 as a new species in the family Iflaviridae is currently under review by the corresponding study group of the International Committee on Taxonomy of Viruses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Carrillo-Tripp, J., Krueger, E. N., Harrison, R. L., Toth, A. L., Miller, W. A. & Bonning, B. C. (2014). Lymantria dispar iflavirus 1 (LdIV1), a new model to study iflaviral persistence in lepidopterans. J Gen Virol 95:2285-2296.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Liu, S., D. Vijayendran, J. Carrillo-Tripp, W.A. Miller, B.C. Bonning. 2014 Analysis of new Aphid lethal paralysis virus isolates suggests evolution of two ALPV species. J. Gen. Virol. 95:2809-19.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Researchers with interests in insect physiology, insect virology and insect pest management. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Jimena Carrillo-Tripp, a postdoctoral research associate, and Diveena Vijayendran, a graduate student have both received training during the course of this project. How have the results been disseminated to communities of interest? Members from the lab presented and discussed their research results at the Keystone meeting on RNA Interference in January, the All Iowa Virology Symposium in March, the annual meeting of the American Society for Virology in July and the annual meeting of the International Society for Invertebrate Pathology in August. What do you plan to do during the next reporting period to accomplish the goals? Characterization of all three viruses will be completed, and results published.
Impacts
What was accomplished under these goals?
During this reporting period we continued the analysis of two aphid viruses and a new gypsy moth virus that was unexpectedly discovered in an insect cell line. The prevalence of the virus ALPV-AP1 in the pea aphid population was 65%. The virus was localized to the midgut epithelium. An additional four insect cell lines were screened for their ability to support replication of ALPV-AP1: a mosquito cell line, C6/36 (Aedes albopictus), a vinegar fly cell line, S2 (Drosophila melanogaster), a wasp cell line, Tex-2 (Trichogramma exiguum) and a spotted cucumber beetle cell line, DU182A (Diabrotica undecimpunctata). When ALPV-AP1 RNA was added to the cell lines, only the wasp (Tex-2) and spotted cucumber beetle (DU182A) cell lines showed changes in cell shape and number, which are indicative of virus infection (cytopathic effects). However, no increase in virus titer was detected in the Tex-2 cell line. A 60-fold increase in virus titer three days after addition of ALPV-AP1 RNA to the DU182A cell line was detected indicating that this cell line can support replication of ALPV-AP1. Aphis glycines virus (AGV) was purified from soybean aphids and the 4.7 kb genome sequence determined. The icosahedral virions are 30 nm in diameter. In contrast to ALPV-AP1, this virus appears to be 100% vertically transmitted. There was no evidence for integration of viral sequences into the soybean aphid genome. In addition to laboratory colonies, the virus was detected in field collected aphids from Iowa, Ohio and Michigan indicating that AGV is widely distributed in the Midwest. LyDiV, discovered in the IPLB-Ld652Y cell line, was named Lymantria dispar virus (LyDiV: Iflaviridae). On the basis of sequence similarity, LyDiV is similar to Deformed wing virus of honey bees, Varroa destructor virus of mites and Formica exsecta iflavirus of ants. As the sequence identities between LyDiV structural proteins and those of its closest relatives are less than 90%, LyDiV can be considered a new virus species. LyDiV replication in IPLB-LD-652Y cells is constrained by RNA interference, an antiviral defense pathway. We screened different cell lines derived from the gypsy moth and found that IPLB-Ld65 and IPLB-LdFB are not infected with LyDiV. The LyDiV-free cell lines will be used to test for LyDiV infectivity. Further analysis of this virus will indicate whether it has potential for use in management of the gypsy moth, a serious pest in U.S. forests. Impact: Aphid viruses have potential for use in management of pestiferous aphids, which cause considerable economic loss by reducing crop yields in temperate climates. Identification of a cell line that supports replication of ALPV-AP1 may facilitate production of this virus for use in suppression of damaging aphid populations. The accidental discovery of the virus LyDiV in one of the cell lines used in this research may provide for a novel approach for suppression of gypsy moth populations which impact forests.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Bonning, B.C., Pal, N., Liu, S., Wang, Z., Sivakumar, S., King, G.F., Miller, W.A. Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids. Nature Biotech December 8, 2013. DOI 10.1038/nbt.2753.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Bonning, B.C., Chougule, N.P. 2013. Delivery of intrahemocoelic peptides for insect pest management. Trends in Biotechnology. Online 12-11-13.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Vijayendran, D., Airs, P.M., Dolezal, K., Bonning, B.C. Arthropod viruses and small RNA. 2013. J. Invertebr. Pathol. 114:186-95.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: During this reporting period we continued with analysis of two aphid viruses and unexpectedly discovered a new gypsy moth virus that was covertly infecting an insect cell line. Having identified two Aphid lethal paralysis (ALPV)-like viruses from pea aphids, we conducted further characterization of ALPV-AP1. We examined virion structure by transmission electron microscopy and the subcellular localization of ALPV-AP1 using silver enhanced immunogold light microscopy. We determined the vertical transmission rate of the virus by collecting newborn nymphs for establishment of single families, and testing these families for the presence of virus by qRT-PCR. A virus-free pea aphid colony was established and aphids from this colony were used as negative controls for these experiments. For analysis of dicistrovirus biology and for production of dicistroviruses for use as insect pest control agents, a cell line that supports virus replication is extremely valuable. Multiple insect cell lines were screened to identify a cell line that will support ALPV-AP1 replication. RNA extracted from purified virions was used to transfect various hemipteran and lepidopteran cell lines. The cell lines were observed for cytopathic effects and tested for increasing amounts of viral RNA. On screening insect cell lines for their ability to support replication of a different dicistrovirus, we tested the cell line IPLB-Ld652Y, which is derived from the gypsy moth, Lymantria dispar, and is commonly used to study insect virus-host interactions. Interestingly, we found another virus of similar shape and size to the dicistrovirus under investigation. We characterized the morphology and genome sequence of the new virus, Lymantria dispar virus 1 or LyDV1. Following on from the discovery of a new virus from the soybean aphid, Aphis glycines virus (AGV), soybean aphid small RNA was sequenced to examine the potential role of the aphid RNA interference pathway in anti-viral defense. Total RNA was isolated from AGV infected soybean aphids. sRNA libraries were prepared from the RNA samples and the sRNA libraries were sequenced using Illumina Genome analyzer II. Soybean aphid small RNA reads were mapped to the AGV genome. A Perl program which allows use of sRNA reads of any length and does not allow mismatched bases, was designed for sRNA sequence mapping. sRNA reads ranging from 10-39 nt were used for the data analysis. During the course of this work, one postdoctoral research associate and one graduate student were trained. Events: Members from the lab presented and discussed their research results at the Keystone meeting on Gene Silencing by Small RNAs, held in Vancouver, Canada, and at the annual meeting of the American Society for Virology, held in Madison, WI. PARTICIPANTS: Bryony Bonning, Principal investigator; Sijun Liu, Assistant Scientist; Jimena Carrillo-Tripp, Postdoctoral Research Associate; Diveena Vijayendran, Graduate Student. Collaborators: W. Allen Miller, Iowa State University; David Murhammer, University of Iowa, IA. TARGET AUDIENCES: Researchers with interests in insect physiology, insect virology, biological control and insect pest management PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Characterization of ALPV-AP1 revealed that this virus has a single stranded RNA genome of 9,888 nt in length. The virus forms an icosahedral particle with a diameter of about 27 nm. Unusually, no vertical transmission of ALPV-AP1 was detected. Infection of uninfected pea aphids by ALPV-AP1 was achieved by microinjection or by feeding on artificial diet containing BSA. In contrast to the many dicistroviruses that are restricted to the gut, tissue specific RT-PCR and immunogold light microscopy analysis of the tissue tropism of ALPV-AP1 indicated that this virus is present in the gut, fat body and bacteriocytes of infected pea aphids. The cell lines derived from Glassy winged sharp shooter (Homalodisca vitripennis; Hemiptera), Leafhopper (Agallia constricta; Hemiptera), Fall armyworm (Spodoptera frugiperda; Lepidoptera) and Cabbage looper (Trichoplusia ni; Lepidoptera) did not support replication of ALPV-AP1. Additional cell lines are being tested. The new discovered virus, LyDV1, which was isolated from the IPLB-Ld652Y cell line has icosahedral virions of approximately 30 nm in diameter. LyDV1 increases in abundance when cells are stressed. The 10 kb polyadenylated RNA genome of LyDV1 has a predicted 0.9 kb 5' UTR, an ORF of 8.9 kb and a 3' UTR of 0.2 kb. The predicted polyprotein of nearly 3,000 amino acids shares highest identity (36 and 37%) with the polyproteins of Varroa destructor virus and Deformed wing virus in the Iflaviridae in the Picornavirales. These results provide a cautionary tale for virologists using cell lines that may contain covert viral infections. Such viruses may alter cellular conditions or express trans-acting viral proteins that affect replication of challenging viruses leading to misinterpretation of results. Our work also further demonstrates the abundance and ubiquity of picorna-like viruses. On analysis of around 14 million small RNA reads from the soybean aphid, the distribution of virus-derived small RNA that mapped to the AGV genome showed a distinct peak at 22 nt. Approximately 8,000 22 nt small RNA mapped to the plus strand (genomic RNA) and 5,000 mapped to the minus strand (replicative intermediate) of the AGV genome. This result indicates that the AGV genome is degraded by the soybean aphid RNA interference pathway. This is the first demonstration that RNA interference is functional in aphid anti-viral defense. Products: The complete sequence of the new viruses ALPV-AP1 and LyDV1 were obtained. Impact: ALPV-AP1 which may have potential for use in aphid management was characterized. Unexpectedly, a new virus of the gypsy moth, LyDV1 was identified and was subsequently characterized. Further analysis of this virus will indicate whether it has potential for use in management of the gypsy moth, a serious pest in U.S. forests. Our research on AGV provides the first evidence that the RNA interference pathway in aphids functions in anti-viral defense.
Publications
- Liu, S., Chougule, N.P., Vijayendran, D., Bonning, B.C. 2012. Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts. PlosOne 7(9): e45161.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities: Having identified a new soybean aphid virus, called Aphis glycines virus (AGV), we continued with work to characterize this virus. In addition we used methods for identification of additional viruses in aphids and in other organisms. Specifically, the small RNA (sRNA) of four aphid species was sequenced. sRNA sequences provide a "fingerprint" that can be used for virus identification and provide information about the regulation of aphid genes. Small RNAs were isolated from Iowa and Ohio colonies of the soybean aphid, and laboratory colonies of pea aphid, green peach aphid, and bird cherry-oat aphid. The sRNA samples were sequenced by using next generation sequencing technology. For each sample, 18-24 million sRNA sequences were obtained. The sequencing data were analyzed with various tools to identify non-coding RNA including microRNA (miRNA), and small interfering RNA (siRNA). The siRNA sequences were also assembled with genome mapping and de novo assembly programs to identify potential new viruses. We also analyzed EST data of a European isolate of the pea aphid and an EST library of the Western corn rootworm (WCR) to look for viral sequences. During the course of this work, one postdoctoral research associate and one graduate student were trained. Events: Members from the lab presented and discussed their research results at the biennial Iowa State University Aphid Research Symposium in February, the annual meeting of the American Society for Virology in July, the Hemipteran-Plant Interactions Symposium in Brazil in July, the annual meeting of the Society for Invertebrate Pathology, held in Halifax, Canada in August, the International Symposium for Molecular Insect Science in Amsterdam, the Netherlands in October, and the annual meeting of the American Entomological Society in November. PARTICIPANTS: Bryony Bonning, Principal investigator; Jimena Carrillo-Tripp, postdoctoral research associate; Diveena Vijayendran, graduate student. Collaborators: W. Allen Miller, Iowa State University; David Murhammer, University of Iowa, IA. TARGET AUDIENCES: Researchers with interests in insect physiology, insect virology and insect pest management PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The newly identified virus AGV has a single stranded RNA genome of 5 kb, and forms 30 nm particles. AGV appears to be transmitted to all offspring and has also been detected in laboratory colonies of two other aphid species, the bird cherry-oat aphid and the green peach aphid. More than 100 different miRNA were identified from sequencing the sRNA of the five aphid samples. The majority of the miRNA were conserved with insect miRNA, with 12 miRNA that appear to be aphid specific. Analysis of sRNA reads, and EST sequences resulted in identification of two Aphid lethal paralysis virus (ALPV)-like viruses (ALPV-AP1, and ALPV-AP2) from pea aphids, a Myzus persicae virus-like virus from the green peach aphid, and an ALPV-like virus from WCR. These results indicate that ALPV-like viruses are widespread across multiple insect orders. Products: The complete sequence of a new ALPV-like virus was obtained. Impact: Through this work, knowledge of aphid viruses, their characteristics and distribution between different insect orders has increased. The next step will be to determine how virulent these viruses are against aphids to assess whether any can be used for management of pestiferous aphids. This is important because aphids are pervasive in temperate agriculture and aphid-related crop damage that results from direct feeding by the aphid, and from aphid transmission of plant viruses, results in considerable economic loss.
Publications
- Giri, L., Feiss, M.G., Bonning, B.C., Murhammer, D.W. 2012. Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. J. Gen. Virol. 93: 389-399.
- Sparks, W.O., Rohlfing, A., Bonning, B.C. 2011. A peptide with similarity to baculovirus ODV-E66 binds the gut epithelium of Heliothis virescens and impedes infection with Autographa californica multiple nucleopolyhedrovirus. J. Gen. Virol. 92:1051-1060.
- Liu, S., Vijayendran, D. and Bonning, B.C. 2011. Next Generation Sequencing Technologies for Insect Virus Discovery. In Insect Viruses, special issue of online journal Viruses 3 (10): 1849-1869 doi:10.3390/v3101849
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: Potential of viruses for aphid control: RNA was isolated from soybean aphids caught in Iowa and short-read sequencing (75 nt) conducted. Sequences (8 million per sample) were analyzed for the presence of viral sequences. Methods for isolation of virus particles from field caught aphids were optimized for extraction of full length genomic RNA from purified virus particles. Achieving this goal will facilitate sequencing of the viral genome. Infection of the pea aphid with Aphid lethal paralysis virus (ALPV) was examined. During the course of this work, two postdoctoral research associates and two graduate student were trained. Events: Members from the lab presented and discussed their research results at the annual meeting for the Society for Invertebrate Pathology, held in Trabzon, Turkey in July, at the All Iowa Virology symposium held in Iowa City, IA in November, and at the annual meeting of the Entomological Society of America, held in San Diego, CA in December. PARTICIPANTS: Bryony Bonning, Principal investigator; Liljana Georgievska and Jimena Carrillo-Tripp, postdoctoral research associates; Wendy Sparks and Diveena Vijayendran, graduate students. Collaborators: W. Allen Miller, Iowa State University; Robert L. Harrison, USDA ARS, Beltsville, MD; David Murhammer, University of Iowa, IA. Training: This project resulted in the training of two postdoctoral research associate, and two graduate students. TARGET AUDIENCES: Researchers with interests in insect physiology and insect pest control. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
RNA sequencing of the soybean aphid resulted in identification of sequences derived from four different viruses: These sequences included those of Rhopalosiphum padi virus (RhPV)-like and Aphid lethal paralysis virus(ALPV)-like viruses, along with a newly identified virus in a distinct, new viral group. This new virus has been called "Aphis glycines virus" (AgV). The fourth sequence was for a "baculovirus repeated ORF" (bro) gene. However, as similar sequences have been found in bacteriophages, in addition to the insect-infecting baculoviruses, this sequence was not investigated further. Classical methods used for virus purification from soybean aphids resulted in isolation of virions of the ALPV-like virus. Sequencing of the genome of this virus is underway. The virus appears to have 80% sequence identity to ALPV. ALPV was shown to infect and replicate in the pea aphid resulting in a significantly reduced lifespan and paralysis of infected aphids. The gut appeared to be the primary site of ALPV replication within the aphid. Products: A new virus of aphids was identified and sequencing of the complete genome is underway. Impact: Identification of new viruses of aphids, and characterization of the virulence of all aphid viruses will facilitate identification of aphid viruses that can be used for aphid management.
Publications
- Giri, L., Li, H., Sandgren, D., Feiss, M.G., Roller, R., Bonning, B.C., Murhammer, D.W. 2010. Removal of transposon target sites from the AcMNPV fp25k gene delays, but does not prevent, accumulation of the few polyhedra phenotype. J. Gen. Virol. 91:3053-64
- Sparks, W.O., Harrison, R.L., Bonning, B.C. 2011. Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut. Virology 409:69-76
- Miller, W.A. and Bonning, B.C. 2010. Dicistroviruses. Annu. Rev. Entomol. 55:129-150
- Johnson, K.N. and Bonning, B.C. 2010. Dicistroviridae. In: Insect Virology, Horizon Scientific Press.
- Harrison, R.L. and Bonning, B.C. 2010. Proteases as insecticidal agents. Special issue of the online journal Toxins, Protein Toxins as Proteases 2: 935-953
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities: Larvae infected with a virus lacking the gene encoding ODV-E56, but expressing GFP were examined for infection of the gut epithelial cells. Following identification of three short peptides with similarity to the ODV-E66 sequence that bind to the H. virescens gut by screening of a phage display library, synthetic peptides were constructed with fluorescent labels. The gut binding activity and ability of these peptides to compete with baculoviruses for binding in the H. virescens gut were assessed. To optimize the infectivity of the infectious clone of Rhopalosiphum padi virus, additional, nonviral bases at the 5' and 3' end of the genome were removed. The susceptibility of several aphid species to infection by RhPV and the related virus Aphid lethal paralysis virus, was tested. During the course of this work, two postdoctoral research associates and one graduate student were trained. Events: Members from the lab presented and discussed their research results at the annual meeting for the Society for Invertebrate Pathology, held in Park City, Utah in August, 2009, the annual meeting of the Entomological Society of America held in Indianapolis in December, 2009 and at an Iowa State University Aphid Research Symposium held in January 2009. Products: New information was gained about baculovirus interaction with the the host gut epithelium, and the of ODV proteins in that process. PARTICIPANTS: Bryony Bonning, Principal investigator; Quiling Li and Liljana Georgievska, postdoctoral research associates; Wendy Sparks, graduate student. Collaborators: Dr. W. Allen Miller, Dr. Xiulian Sun. This project resulted in the training of two postdoctoral research associates, and one graduate student. TARGET AUDIENCES: Researchers with interests in insect physiology and insect pest control. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Fluorescence microscopy showed that GFP expression was absent from midgut epithelial cells infected with the virus lacking odv-e56. Despite binding to and fusing with the cell, the viral DNA did not enter the nucleus of the midgut epithelial cell where GFP expression would have been initiated. Synthetic peptides with fluorescent labels were used to confirm peptide binding to the midgut of H. virescens. These peptides, but not the negative control peptide, also bound to the guts of other insect species. In competition assays one the peptides decreased mortality of larvae at an LD50 dose. Oral administration of this peptide also increased the survival time of larvae fed higher doses of virus. These results suggest that ODV-E66 is important for initial virus-host interaction in the gut. Additional sequences at the 5' and 3' end of the genome of the infectious clone of RhPV which could negatively impact infectivity were removed. Infection of the pea aphid Acyrthosiphon pisum (Harris), with ALPV was confirmed. Infected aphids were paralysed and fell from the host plant. The presence of negative strand RNA confirmed virus replication. Impact: Increased knowledge of the interaction of a baculovirus with the insect gut may in the long term allow for genetic manipulation of baculovirus host range. This would be a particularly useful tool for management of multiple pests that are not all equally susceptible to a baculovirus. Optimization of the infectivity of the infectious clone of RhPV will improve the usefulness of this tool for understanding fundamental and applied aspects of dicistrovirus biology. Defining the host range of the aphid dicistroviruses will provide insight for their potential use as biological control agents
Publications
- Schmidt, N.R. and Bonning B.C. 2009. Intrahemocoelic toxins for lepidopteran pest management. In Molecular Biology and Genetics of Lepidoptera (M. Goldsmith, F. Marc, Eds). Taylor and Francis.
- B.C. Bonning. 2009. The Dicistroviridae: An emerging family of invertebrate viruses. Virologica Sinica 24 (4): 417-427.
- Sun, X., Wu, D., Sun, X., Jin, L., Ma, Y., Bonning, B.C., Peng, H., Hu, Z. 2009. Impact of Helicoverpa armigera nucleopolyhedroviruses expressing a cathepsin L-like protease on target and nontarget insect species on cotton. Biol. Control 49 (1): 77-83.
- Schmidt, N.R., Haywood, J.M., Bonning, B.C. 2009. Toward the physiological basis for increased Agrotis ipsilon multiple nucleopolyhedrovirus infection following feeding of Agrotis ipsilon larvae on transgenic corn expressing Cry1Fa2. J. Invertebr. Pathol. 102(2): 141-8
- Harrison, R.L., Sparks, S., Bonning, B.C. 2010. The Autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be functionally substituted by the Rachiplusia ou multiple nucleopolyhedrovirus ODV-E56. J. Gen Virol. doi:10.1099/vir.0.017160-0
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities: The role of the baculovirus occlusion-derived envelope protein, ODV-E56, was evaluated by binding and fusion assays, and by injection into the tobacco budworm, Heliothis virescens. Gut proteins of the host insect were examined to identify proteins that bind to ODV-E56. A phage display library was screened for analysis of peptides that bind to the H. virescens gut. ScathL, a cathepsin L-like cysteine protease, functions in basement membrane remodeling during insect development. The physiological impact of baculovirus expression of ScathL in H. virescens and the pea aphid, Acyrthosiphon pisum was assessed. For investigation of the replication and pathogenesis of dicistroviruses, a newly characterized virus family, we constructed clones of the aphid virus Rhopalosiphum padi virus (RhPV) to test for infectivity in the insect cell line GWSS-Z10 and in aphids. During the course of this work, two research associates, and three graduate students were trained. Events: Members from the lab presented and discussed their research results at the annual meeting of the Society for Invertebrate Pathology held in Warwick, UK in August 2008, the annual meeting of the Kansas Entomological Society, and the All Iowa Virology conference held in September 2008. Products: New fundamental knowledge was acquired about the role of ODV proteins in baculovirus - insect host interaction, and the physiological impact of ScathL on the host insect. PARTICIPANTS: Participants: Bryony Bonning, Principal investigator; Huarong Li, postdoctoral research associate, and Liljana Georgievska, predoctoral research associate; Nina Schmidt, Wendy Sparks and Hailin Tang, graduate students. Collaborators: Dr. John Gatehouse, Dr. W. Allen Miller. Training: This project resulted in the training of two research associates, and three graduate students. TARGET AUDIENCES: Researchers with interests in insect physiology, insect viruses, and insect pest control. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A null mutant baculovirus lacking the gene encoding ODV-E56, was characterized. ODV-E56 is required for oral infectivity but not for infectivity following injection of virus into the host. There was no significant difference in binding and fusion of virus lacking ODV-E56 and wild type virus suggesting that the block to virus infectivity occurs downstream of the initial binding and fusion events. A protein of unknown identity from the gut of the host insect bound ODV-E56. Screening of a phage display library for peptides that bind to the H. virescens gut resulted in isolation of three gut binding peptides with identity to another ODV protein, ODV-E66 from three different species of nucleopolyhedroviruses. This result suggests that ODV-E66 is important for initial virus-host interaction in the gut. Larvae of H. virescens infected with a recombinant baculovirus melanize (turn black) prior to death. We showed that melanization was closely associated with the high cysteine protease activity of ScathL in the hemolymph, but not with the presence of a catalytic site mutant of ScathL produced by a control baculovirus. ScathL expression resulted in fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in larvae infected with the ScathL-expressing baculovirus. The median lethal dose for purified ScathL injected into fifth-instar H. virescens was 11.0 microgram per larva. ScathL was also lethal to adult pea aphids, with a similar loss of integrity of the gut and fat body. Injection with the catalytic site mutant of ScathL, or bovine trypsin at 20 microgram per larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity. We constructed the first infectious clone of a dicistrovirus, RhPV and showed that RNA transcribed from the cDNA clone is infectious to the insect cell line GWSS-Z10. Infection resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from the infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool for the study of dicistroviruses. Impact: Understanding the interaction of a baculovirus with the insect gut may in the long term allow for genetic manipulation of baculovirus host range. This would be a particularly useful tool for management of multiple pests that are not all equally susceptible to a baculovirus. The use of the protease ScathL for pest management purposes will be facilitated by understanding of the exact mechanism of insecticidal action of this protease. The infectious clone of RhPV provides a unique and valuable tool for analysis of fundamental dicistrovirus biology and will also facilitate potential use of this virus for aphid control.
Publications
- Sparks, W.O., Bartholomay, L. and Bonning B.C. 2008. Insect Immunity to Viruses. In: Insect Immunology, N.E. Beckage, Ed. Academic Press 209-242.
- Federici, B.A., Bonning, B.C. and St Leger, R. 2008. Improvement of insect pathogens as insecticides through genetic engineering. In. PathoBiotechnology. Colin Hill and Roy Sleator, Eds. Landes. Chapter 2, pp. 15-40
- Sparks, W., Li, H., Bonning, B.C. 2008. Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus. Journal of Visualized Experiments (19). pii: 888. doi: 10.3791/888.
- Li, H., Sparks, W., Bonning, B.C. 2008. Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus. Journal of Visualized Experiments (18). pii: 889. doi: 10.3791/889.
- Boyapalle, S., Beckett, R.J., Pal, N., Miller, W.A. and Bonning, B.C. 2008. Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone. Virology 375(2): 401-411.
- Li, H., Tang, H., Sivakumar, S., Philip, J., Harrison, R.L. Gatehouse, J.A. and Bonning, B.C. 2008. Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris). Journal of Insect Physiology 54(5): 777-789.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: The role of baculovirus occlusion-derived virus (ODV) protein ODV-E56 was evaluated by gene disruption and the impact of odv-e56 disruption on virulence assessed by bioassay. To elucidate the insecticidal mechanism of action of the basement membrane degrading protease, ScathL, baculoviruses co-expressing ScathL and the reporter enzymes CAT or beta-galactosidase were constructed. The expression of these enzymes was used to follow the course (lacZ) and extent (CAT) of baculovirus infection and to assess whether ScathL altered the course of virus infection in any way. The specificity of ScathL proteolytic degradation was assessed by scanning and transmission electron microscopye analysis of insects infected with viruses expressing ScathL, or the catalytically inactive control protein ScathLC146A. In vitro assessment of degradation of dissected basement membrane was also conducted with degradation assessed by separation of proteins in denaturing polyacrylamide gels.
During the course of this work, one postdoctoral research associate and two graduate students were trained. Events: Members from the lab presented and discussed their research results at the annual meeting for the Society for Invertebrate Pathology, held in Quebec, Canada in August, 2007. Products: New fundamental knowledge was acquired relating the function of baculovirus ODV proteins and the mechanism of insecticidal action of ScathL. A collaboration was established with Dr. John Gatehouse, University of Durham, UK for work on the biochemistry of ScathL.
PARTICIPANTS: Bryony Bonning, Principal investigator; Huarong Li and Narinder Pal, postdoctoral research associates; Sandhya Boyapalle, Wendy Sparks and Hailin Tang, graduate students. Collaborators: Dr. John Gatehouse, Dr. W. Allen Miller. Training: This project resulted in the training of two postdoctoral research associate, and three graduate students.
TARGET AUDIENCES: Target audiences: Researchers with interests in insect physiology and insect pest control. Efforts: This project provided three graduate students with research training.
Impacts
A null mutant baculovirus lacking the odv-e56 gene was constructed. Bioassays of this virus along with wild type virus and a control revertant virus in Heliothis virescens demonstrated that odv-e56 is essential for oral infectivity and hence is a newly characterized pif (per oral infectivity) gene. It was not possible to construct a null mutant lacking the odv-e18 gene which suggests that this gene may be essential for virus viability. The mechanism of insecticidal action of the basement membrane-degrading protease ScathL was investigated. We determined that baculovirus expression of ScathL did not result in alteration of the course of virus infection through disruption of the basement membrane. Rather, damage to the basement membrane itself appears to be sufficient for the insecticidal impact. Tissue specificity analysis confirmed that ScathL has high specificity for the basement membrane at low doses. It was unclear whether disruption of other tissues at high doses
resulted from direct proteolytic action of ScathL, or from the loss of tissue integrity as a result of disruption of the overlying basement membrane. Impact: Understanding the interaction of a baculovirus with the insect gut may in the long term allow for genetic manipulation of baculovirus host range. This would be a particularly useful tool for management of multiple pests that are not all equally susceptible to a baculovirus. The use of the protease ScathL for pest management purposes will be facilitated by understanding of the exact mechanism of insecticidal action of this protease.
Publications
- Bonning, B.C. and Nusawardani, T. 2007. Introduction to the use of baculoviruses as biological insecticides. In: Baculovirus and Insect Cell Expression Protocols (D. Murhammer, Ed.), Humana Press, Totowa, New Jersey. Chapter 24. pp. 359-366.
- Li, H. and Bonning, B.C. 2007. Evaluation of the insecticidal efficacy of wild type and recombinant baculoviruses. In: Baculovirus and Insect Cell Expression Protocols (D. Murhammer, Ed.), Humana Press, Totowa, New Jersey. Chapter 20, pp. 379-404.
- Boyapalle, S., Pal, N., Miller, W.A., Bonning, B.C. 2007. A glassy-winged shartpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae). J. Invertebr. Pathol. 94(2): 130-139.
- Li, H., Tang, H., Harrison, R.L. and Bonning, B.C. 2007. Impact of a Basement Membrane-Degrading Protease on Dissemination and Secondary Infection of Autographa californica Multiple Nucleopolyhedrovirus in Heliothis virescens L. J. Gen Virol. 88: 1109-1119.
- Philip, J., Fitches, E., Harrison, R. L., Bonning, B.C. and Gatehouse, J.A. 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Molec. Biol. 37(6): 589-600.
- Pal, N., Boyapalle, S., Beckett, R., Miller, W.A. and Bonning, B.C. 2007. A baculovirus-expressed dicistrovirus that is infectious to aphids. J Virol. 81 (17):9339-45
- Tang, H., Li, H., Lei, S., Harrison, R.L. and Bonning, B.C. 2007. Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens. Tissue and Cell 39: 431-443
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Progress 01/01/06 to 12/31/06
Outputs
Peptides that bind to the gut of Heliothis virescens share sequence homology with a protein present in baculovirus occlusion-derived virus (ODV), indicating that this protein may play a pivotal role in baculovirus binding to the insect gut. An insect cell line (GWSS Z10) that supports replication of Rhopalosiphum padi virus (RhPV) has been identified. This cell line can be used for further study of the biology of RhPV. An infectious clone of RhPV has been constructed and infectivity of the clone to both aphids and the cell line demonstrated. Use of the baculovirus expression system for production of infectious RhPV in a cell line that does not support RhPV replication has also been completed.
Impacts
Understanding the interaction between a baculovirus and its insect host will facilitate optimization of baculoviruses for use in insect pest management. Acquisition of an infectious clone of RhPV and identification of a cell line that supports RhPV replication provide the necessary tools for analysis of RhPV biology and elucidation of the potential of this virus for pest management purposes.
Publications
- Liu, S., Li, H., Sivakumar, S. and Bonning, B.C. 2006. Virus-derived genes for insect resistant transgenic plants. In. Advances in Virus Research, vol. 68 Insect Viruses: Biotechnological Applications, pp 428-457.
- Advances in Virus Research: Insect Viruses: Biotechnological Applications 2006, volume 68. Editor, B.C. Bonning.
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Progress 01/01/05 to 12/31/05
Outputs
The genes encoding the baculovirus occlusion-derived virus (ODV) envelope proteins odv-e18 and odv-e56 have been cloned for in vitro production of recombinant proteins that will be used for production of antisera. To test the hypothesis that these positively selected proteins play a key role in host specificity, we established assays using brush border membrane vesicles derived from Heliothis virescens to test for binding and fusion of R18-labeled ODV.
Impacts
Examination of the role of the two baculovirus envelope proteins in the initial infection of a host insect will increase our understanding of the evolutionary interactions between the virus and the host insect, and facilitate optimization of baculoviruses for use in insect pest management.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Nine insect cell lines derived from Lepidoptera, Diptera and Homoptera were screened for susceptibility to infection with Rhopalosiphum padi virus (RhPV). Two homopteran cell lines that support replication of RhPV have been identified. An infectious clone of RhPV that replicates in cell culture and in aphids has been isolated. Infection was demonstrated by western blot detection of viral coat proteins, following transfection of RhPV RNA on to insect cells, detection of the negative strand replicative form by reverse transcription PCR in both insect cells and infected aphids, and by northern blot analyses.
Impacts
The infectious clone of RhPV has potential for use in aphid management. Production of novel virus-based strategies for management of insect pests may contribute toward a sustainable agriculture.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
By using gelatin zymography, and azocoll, azocasein, and azoalbumin assay of cells and medium from infected HighFive and Sf21 cell cultures, protease activity was detected for baculovirus expressed XcGV MMP and Dm1-MMP. No active enzyme was detected for MSV176, MSV179, MSV175 or HCB. However, melanization was seen for larvae infected with viruses expressing MSV175, MSV176, MSV179 and XcGV MMP. The titer of the budded virus stock of MSV179 decreased over a two week period, suggesting that this protease degrades a component of the budded virus. Viruses expressing Dm1-MMP and HCB did not show insecticidal effects when tested against neonate larvae of Heliothis virescens. In both cases, the enzymes are expressed as proenzymes. We produced activated forms of Dm1-MMP, and plan to produce an activated form of HCB.
Impacts
Production of novel virus-based strategies for management of insect pests may contribute toward a sustainable agriculture. Use of the baculovirus expression system also allows screening for insect toxins that are active within the body cavity of the insect.
Publications
- No publications reported this period
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