41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

Viral disease is a major component of the cyclic population dynamics of some Lepidoptera including western tent caterpillars. Epizootics of nucleopolyhedrovirus and host population subdivision provide an arena in which selection on virulence of virus and resistance of hosts could act. Theory predicts that epizootics should select for host resistance and that viral isolates should respond to this change on a population-by-population basis. Experiments provide evidence that these interactions are occurring but that patterns are weak as compared to other factors that determine the cyclic population dynamics. In addition there is no evidence for induced immunity or selection within a generation of tent caterpillars. The factors that promote the rapid development of NPV epizootics remain a mystery and are the topic of future research.

Codling moth larvae from 23 orchards located in five European countries were tested for their susceptibility/resistance to the Cydia pomonella granulovirus (CpGV) in standardized laboratory bioassays. In general, the results from the bioassays were in accordance with the observations in the field, i.e. most orchards from which the farmer reported failure of the CpGV treatment contained resistant codling moth populations. This was found in all of the countries investigated. The estimation of the percentage of resistant individuals in resistant populations ranged roughly from 30 to 90%. However, in some apparently susceptible populations there were also hints for the presence of a very small fraction of resistant individuals. Fourteen of these European populations were tested for susceptibility to eight insecticides including different classes of insect growth regulators and neurotoxic compounds. High mortality was recorded to most insecticides, ranging from 86% (azinphos-methyl) to 100% (deltamethrin), independent of resistance to CpGV. A reduced susceptibility to azinphos-methyl, chlorpyriphos-ethyl, spinosad and tebufenozide was recorded in several populations. Overall, there was no indication for the occurrence of cross-resistance between CpGV and insecticides in the tested populations.

Many recent emerging infectious diseases in humans, such as HIV and Ebola resulted from host shifts, are maintained within wildlife populations, and pose a substantial health risk. Most research has focused on controlling epidemics, however, using model systems can formulate predictions about the factors that lead to successful disease emergence. Here, I employ an insect-virus system to test the conditions that lead transient infections to become self-sustaining diseases. The Indian meal moth (Plodia interpunctella) and the Almond moth (Ephestia cautella) are worldwide pests of stored food products, and due to their tractability in laboratory experiments have been used to study host-parasite dynamics. EcNPV, a nucleopolyhedrovirus, is largely host specific on Ephestia, but can be transmitted to the new host, Plodia; demonstrating altered disease expression. PiGV, a granulosis virus, is host specific on Plodia, with little evidence of transmission to Ephestia. Here, I measure how infection route affects the infectivity of each virus on both hosts; testing the standard oral inoculation route versus direct intrahaemocoelic injections of the inclusion-bodied virus on important epidemiological parameters (infectivity, disease induced mortality, sub-lethal effects, covert infection). These findings will elucidate the important components of host-pathogen dynamics that can lead to long-term sustainability of emerging diseases.

Gypsy moth (Lymantria dispar) larvae show intrastadial developmental resistance (IDR) to the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV); newly molted larvae are 40-50% more susceptible to a given dose of virus than those inoculated 48-72 hours post-molt. We hypothesized that there are differences in the immune responses between the larval ages, and they contribute to IDR. We have identified several possible immune processes that may be anti-viral defenses, such as cellular encapsulation, phenoloxidase (PO) mediated melanization, and apoptosis of infected cells. To examine cellular encapsulation, we inserted infected tissue into larvae of different ages and observed the extent of the immune response to the inserts. We also inoculated both susceptible and resistant-aged larvae intrahemocoelically and measured the PO activity in the hemolymph periodically following the inoculation. Finally, we established if apoptosis was occurring in infected tissues by assaying infected trachea for apoptotic cells. From our results, resistant-aged larvae show a greater cellular immune response and exhibit higher PO activity than susceptible-aged larvae. Apoptosis occurs readily in infected trachea of both ages of larvae, but the impact on infection appears to be greater in resistant-aged larvae. These findings indicate the importance of the immune system in IDR, and in the future we will continue to define the mechanisms behind these anti-viral defenses.

Organic soybean is a leading organic crop in the United States that is damaged by infestations of Anticarsia gemmatalis larvae. Because baculoviruses are available to organic farmers for controlling crop pests, we characterized infection and pathogenesis of Autographa californica M nucleopolyhedrovirus (AcMNPV), in A. gemmatalis larvae using an AcMNPV recombinant carrying the lacZ reporter gene (AcMNPV-hsp70lacZ). Newly-molted fifth instar larvae inoculated orally with occlusion bodies (OB) or intrahemocoelically with budded virus (BV) were highly resistant to fatal infection. Dosages of 5300 OB were required to generate LACZ-positive cells in the midgut, but once infection was established, it was efficiently transferred to adjacent tracheoblasts. However, time course studies revealed that infection did not subsequently disseminate throughout the hemocoel. Lack of distal spread from infected tracheoblasts and low systemic infectivity of BV are suggestive of a virus-elicited immune response, but we did not observe hemocytes associated with LACZ-positive cells. Studies using flow cytometry demonstrate that A. gemmatalis hemocytes are resistant to infection by BV, which may explain the low infectivity of AcMNPV. Because neither AcMNPV OB nor BV were highly pathogenic in A. gemmatalis larvae, AcMNPV may not be effective in organic cropping systems for controlling A. gemmatalis larvae.

AcMNPV is the best-studied member of the Baculoviridae family and most of the genes identified in this virus serve as a basis for comparison to other baculoviruses. A single-stranded DNA binding protein, LEF-3 (407 aa, 45 kDa), is essential for AcMNPV (Ac) DNA replication. LEF-3 also transports P143, a helicase, to the nucleus. We predict that LEF-3 has functional domains including ones responsible for ssDNA binding, P143 interaction, and nuclear localization. Site-directed mutagenesis revealed that N-terminal aa 5 to 56 are responsible for nuclear localization (NLS), while aa 2 to 125 are required for P143 interaction. Alignment of type I NPV LEF-3s revealed a region (aa 20-28 in AcLEF-3) not present in all species. Fluorescence microscopy showed that aa 3-48 of CfMNPV (Cf) LEF-3 are sufficient for nuclear import, suggesting that aa 20-28 of AcLEF-3 are not essential for the NLS. Substitution of conserved basic residues with nonpolar residues did not affect nuclear localization. However, combining individual mutations with the deletion of aa 20-28 resulted in cytoplasmic AcLEF-3. This suggests a novel system for nuclear import that may involve the structure of LEF-3. The possibility that AcLEF-3 has developed multiple NLS sequences to enhance replication efficiency is also being considered. A similar approach was used within aa 2-125 to investigate LEF- 3/P143 interaction. Preliminary results show that deleting Gly552 in P143 inhibits interaction with LEF-3. Transient replication assays show that LEF-3 function extends beyond nuclear localization and transport of P143. To further characterize the role of LEF-3 in DNA replication and late gene expression, the ability of various LEF-3 mutants to rescue function in the presence of a knockout bacmid will be reported.

Given the high genetic diversity of insect baculoviruses co-infections are likely to be common in natural populations. The baculovirus life cycle comprises two main components, infection and spread in the host (within-host dynamics) and transmission to naïve hosts (between host transmission); most mixed infection studies have targeted the ‘between host’ component. We investigated within-host mixed infections using an AcMNPV clone lacking an effective ecdysteroid UDP glucosyl transferase (egt) gene. It is well established that deletion of egt results in a more rapid kill, with resultingly lower occlusion body (OB) yield. This is assumed to enhance between host transmission because more inoculum is released into the environment. However, whether the possession of this gene is likely to have a cost or a benefit for within-host infection is not known. The results indicate that dual genotype infection parameters can be predicted from single infections, in a qualitative sense, for speed of kill, and to a lesser extent, virus yield. Considerable variation in the genotype ratio was observed between individual larvae; possibly as a result of a small number of virions initiating infection. Longer term passage experiments showed that there was selection for the parent wild type genotype over the egt deletion strain.

The cabbage looper, Trichoplusia ni, has become a serious pest of greenhouse vegetable production in Canada due in part to the development of resistance to Bacillus thuringiensis (Bt) based bioinsecticides. Most Canadian greenhouse vegetable production is chemical pesticide free, relying on biological control agents deployed in an IPM system for insect pest control, thus alternatives to Bt products are required. Indigenous strains of TnSNPV and AcMNPV were selected based on laboratory assessments of infectivity and virulence. Selected strains were evaluated as microbial pesticides in a series of cage and open compartment greenhouse spray trials on cucumber plants artificially infested with T. ni larvae. Preliminary dose uptake spray trials with 2nd and 4th instar larvae showed high levels of infection and mortality with both TnSNPV and AcMNPV at dose levels ranging from 5.0x1010 to 1.0x1012 OB/Ha equivalents (400L/Ha). Subsequent caged trials demonstrated greater than 90% mortality in 2nd instar populations by nine days post treatment for both TnSNPV and AcMNPV isolates. Finally, a larger scale spray trial was conducted on cucumber plants, seeded with 2nd instar T. ni, sprayed with the AcMNPV isolate at 1.0x1012 OB/Ha (400L/Ha volume equivalent) and destructively sampled at 2, 6, 12 and 14 days post treatment. The AcMNPV treated plots showed significantly lower numbers of surviving larvae and lower foliage and fruit damage at 6 days post treatment and onwards. These initial results indicate good potential for indigenous baculovirus isolates as biological control agents of T. ni in greenhouse vegetable production.

MicroRNAs (miRNAs) are a class of small, RNAs found in plants, animals and viruses capable of post-transcriptional regulation of specific mRNAs by inhibiting their translation. A number of different viruses including Herpes viruses and SV 40 have been shown to code for miRNAs. Most viral miRNAs are contained within a ~200 nt primary miRNA transcript which is processed into a ~ 75 nucleotide long pre-miRNA domain capable of forming a stem-loop (hairpin) structure with a calculated minimal folding free energy less than -25kcal/mol. These pre-miRNAs are then cleaved into a ~22 nt long single stranded active miRNA. Here we report on our analysis of the genomes of four insect pathogenic, DNA viruses for the presence of putative miRNA sequences. The salivary gland hypertrophy virus (SGHV) of tsetse flies was found to contain a total of 7 putative miRNAs and the genome of the closely related house fly virus, MdSGHV had 6 putative miRNA sequences. AcMNPV was found to have the fewest miRNAs with only 4 of these sequences present while Hz-2V contains 14 putative miRNAs. The sequence and location of these miRNAs is presented as well as the possible role they might play in the pathology of these viruses.

Baculoviruses produce both overt and covert infections in lepidopteran hosts. In the covert or persistent infections, which are vertically transmitted between generations it is unclear if the virus DNA exists in an integrated state within the host genome, as an episome or within virus particles. Our lack of understanding of the physical status of covert baculovirus genomes stems partly from their very low level within the insect. Although they can be detected using polymerase chain reaction (PCR) and quantitative (Q) PCR they are often at the limits of reliable detection. Persistent baculovirus infections in laboratory insects pose serious problems since they may reactivate spontaneously or be triggered by a heterologous baculovirus infection. To characterize persistent baculovirus genomes further we have isolated total DNA from M. brassicae, T. ni and S. exigua insects and fractionated it using sucrose velocity gradients. Virus DNA either co-migrated or migrated apart from cellular DNA in an insect species-dependent manner. To characterize the virus DNA from sucrose gradient fractions further we amplified it in vitro using random primers with DNA polymerase. This amplified DNA could then be readily analysed using PCR to produce products that were sequenced to confirm the identity of the viruses. DIG-labelled probes were also derived from the amplified DNA. These were used to probe filters containing restriction enzyme digests of bacmids to determine if complete genome sequences were present in the original virus DNA isolated from persistent infections. These results suggested that there may be differences between the genomes of persistent viruses and their wild type counterparts.