Lymantria Dispar Multiple Nucleopolyhedrovirus Research Articles

Genome Sequencing and Organization of Three Geographically Different Isolates of Nucleopolyhedrovirus from the Gypsy Moth Reveal Significant Genomic Differences.

The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of trees and forests. Lymantria dispar nucleopolyhedrovirus (LdMNPV) belongs to the Baculoviridae family and is an insect virus specific to gypsy moth larvae. In this study, we describe the complete genome sequences of three geographically diverse isolates, H2 (China), J2 (Japan), and T3 (Turkey), of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). The genomes of isolates H2, J2, and T3 were subjected to shotgun pyrosequencing using Roche 454 FLX and assembled using Roche GS De Novo Assembler. Comparative analysis of all isolates was performed using bioinformatics methods. The genomes of LdMNPV-H2, J2, and T3 were 164,746, 162,249, and 162,614 bp in size, had GC content of 57.25%, 57.30%, and 57.46%, and contained 162, 165, and 164 putative open reading frames (ORFs ≥ 150 nt), respectively. Comparison between the reference genome LdMNPV-5/6 (AF081810) and the genomes of LdMNPV-H2, J2, and T3 revealed differences in gene content. Compared with LdMNPV-5/6, ORF5, 6, 8, 10, 31, and 67 were absent in LdMNPV-H2, ORF5, 13, and 66 were absent in LdMNPV-J2, and ORF10, 13, 31, and 67 were absent in LdMNPV-T3. In addition, the gene encoding the mucin-like protein (ORF4) was split into two parts in isolates H2 and T3 and designated ORF4a and ORF4b. Phylogenetic analysis grouped isolates H2 and J2 in a different cluster than isolate T3, which is more closely related to the Turkish and Polish isolates. In addition, H2 was found to be closely related to a South Korean LdMNPV isolate. This study provided a more detailed overview of the relationships between different geographic LdMNPV isolates. The results showed remarkable differences between groups at the genome level.

Four antimicrobial peptides of Asian gypsy moth respond to infection of its viral pathogen, nucleopolyhedrovirus (LdMNPV)

Antimicrobial peptides (AMPs) play essential roles in defending against various invading pathogens. Although antibacterial or antifungal properties of AMPs have been well characterized, the contribution of AMPs to immune defenses against viruses especially baculoviruses is still unclear. In this study, four full-length AMP genes (Ldcec, Ldatt, Ldglo and Ldmor) that encode the cecropin, attacin, gloverin and moricin, respectively, were characterized in Lymantria dispar (Asian gypsy moth). All four AMPs were cationic peptides and exhibited hydrophilicity. Structural analysis showed that the Ldcec and Ldmor were α-helical peptides. Tissue-specific Ldcec expression was the highest in fat body, while expression of Ldatt, Ldglo and Ldmor was the highest in epidermis. All four AMP genes were expressed during all developmental stages with the highest expression in the pupa and adult. Compared to mock infection, expression of these four AMP genes were significantly induced following Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) challenge and sharply increased at 72 h post infection. After Ldglo gene silencing, the DNA replication levels of LdMNPV in L. dispar larvae significantly increased at 48 and 72 h post infection, indicating that the Ldglo could suppress the DNA replication of LdMNPV. Our results suggest that four AMPs of L. dispar may play important roles in antiviral immunity against LdMNPV.

The PI3K/AKT Pathway and PTEN Gene Are Involved in "Tree-Top Disease" of Lymantria dispar.

Nucleopolyhedrovirus (NPV) can alter its host behaviour such that infected larvae hang at the top of trees before their death. This phenomenon was firstly described by Hofmann in 1891 and named as “tree-top disease”. Subsequent studies have described effects during the infection proceedings as NPVs manipulate the host to avoid the immune response, cross defensive barriers and regulate hormones. In this study, we demonstrate that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway is involved in host manipulation by Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Particularly at the late stage of infection, a multifunctional dephosphorylase in the PI3K/AKT signaling pathway is dynamically upregulated, namely, the phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) gene. The biological assays of PTEN gene knockdown showed that an increase in PTEN gene expression was necessary for the infected Lymantria dispar larvae’s terminal climbing behavior, death postponement and virion production. The results imply that the PI3K/AKT signaling pathway and PTEN gene might play an essential role in “tree-top disease” induced by LdMNPV.

Identification of Core Genes of Toll-like Receptor Pathway from Lymantria dispar and Induced Expression upon Immune Stimulant.

Simple SummaryThe gypsy moth, Lymantria dispar is one of the main forest defoliators worldwide. The baculovirus, Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), can naturally control this pest and is safe to non-target organisms. However, Toll-like receptor (TLR) signaling pathway, which plays a critical role in innate immunity both in vertebrates and invertebrates, and its underlying immune mechanism against LdMNPV invasion in L. dispar have not been investigated. In this study, eleven TLRs and five downstream TLR pathway components of L. dispar were identified. All of these proteins comprised typical domain architecture. Primary and tertiary structure analysis revealed that Toll/interleukin-1 receptor (TIR) domains of TLRs from L. dispar were conserved during evolution. Expression analysis showed that several TLRs and all identified downstream genes of TLR pathway in L. dispar were significantly up-regulated in response to LdMNPV infection, implying that the TLR pathway of L. dispar was activated and may be involved in L. dispar innate immunity against LdMNPV infection. Taken together, this research contributed to the clarification of innate immunity in L. dispar.The gypsy moth, Lymantria dispar, is a polyphagous forest pest worldwide. The baculovirus, Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) is a natural pathogen of L. dispar. The Toll-like receptors (TLR) pathway plays a crucial role in both innate and adaptive immunity in animals. However, The TLR pathway and its underlying immune mechanism against baculovirus in L. dispar have not been explored. In this study, eleven TLRs and five downstream TLR pathway components were identified and characterized from L. dispar. Structural analysis indicated that intracellular Toll/interleukin-1 receptor (TIR) domains of LdTLRs and LdMyD88 contained three conserved motifs, and the 3D structures of TIR domains of LdTLRs possessed similar patterns in components arrangement and spatial conformation. The TLR proteins of L. dispar were placed into five monophyletic groups based on the phylogenetic analysis. LdTLR1, 2, 5, 6, 7, 8 and all identified downstream TLR pathway factors were highly induced upon LdMNPV infection, indicating that the TLR pathway of L. dispar was activated and might play a role in the immune response to LdMNPV infection. Collectively, these results help elucidate the crucial role of the TLR pathway in the immune response of L. dispar against LdMNPV, and offer a foundation for further understanding of innate immunity of the pest.

Pathology and genome sequence of a Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) isolate from Heilongjiang, China

The pathogenicity and genome sequence of isolate LdMNPV-HrB of the gypsy moth alphabaculovirus, Lymantria dispar multiple nucleopolyhedrovirus from Harbin, Heilongjiang, China, were determined. A stock of this virus from one passage through the gypsy moth New Jersey Standard Strain (LdMNPV-HrB-NJSS) exhibited 6.2- to 11.9-fold greater pathogenicity against larvae from a Harbin colony of L. dispar asiatica than both Gypchek and a Massachusetts, USA LdMNPV isolate (LdMNPV-Ab-a624). Sequence determination and phylogenetic analysis of LdMNPV-HrB and LdMNPV-HrB-NJSS revealed that these isolates were most similar to other east Asian LdMNPV isolates with 98.8% genome sequence identity and formed a group with the east Asian LdMNPV isolates which was separate from groups of isolates from Russia, Europe, and USA.

A Comparison of the Vertical Transmission of High- and Low-Virulence Nucleopolyhedrovirus Strains in Lymantria Dispar L.

Baculoviruses can persist in insect host organisms after infection and may be vertically transmitted to the next generation, in which they may be reactivated. The goal of the present study was to compare the efficiency of the vertical transmission of high- and low-virulence strains and the subsequent reactivation of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) in the offspring of Lymantria dispar L. adults who survived after viral infection. As a result of parental infection, the fecundity of survived females, pupae weight, and fertility were significantly different compared to the untreated insects. However, differences in these parameters between high- and low-virulence strains were not observed. The prevalence of virus strains in the offspring measured by quantitative polymerase chain reaction also did not differ. When the larvae reached the fourth instar, they were starved to activate the vertically transmitted virus. The frequency of virus activation in the experiment was not dependent on the virulence of the virus strains. These results are helpful for understanding the strategy of virus survival in nature and for the selection of the most effective strains with transgenerational effects in the years following pest treatment.

Comparative efficacy of gypsy moth (Lepidoptera: Erebidae) entomopathogens on transgenic blight-tolerant and wild-type American, Chinese, and hybrid chestnuts (Fagales: Fagaceae).

American chestnut (Castanea dentata [Marsh.] Borkh.) was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen, Cryphonectria parasitica (Murrill) Barr, functionally eliminated it across its range. One promising approach toward restoring American chestnut to natural forests is development of blight-tolerant trees using genetic transformation. However, transformation and related processes can result in unexpected and unintended phenotypic changes, potentially altering ecological interactions. To assess unintended tritrophic impacts of transgenic American chestnut on plant-herbivore interactions, gypsy moth (Lymantria dispar L.) caterpillars were fed leaf disks excised from two transgenic events, Darling 54 and Darling 58, and four control American chestnut lines. Leaf disks were previously treated with an LD50 dose of either the species-specific Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) or the generalist pathogen Bacillus thuringiensis subsp. kurstaki (Btk). Mortality was quantified and compared to water blank controls. Tree genotype had a strong effect on the efficacies of both pathogens. Larval mortality from Btk-treated foliage from only one transgenic event, Darling 54, differed from its isogenic progenitor, Ellis 1, but was similar to an unrelated wild-type American chestnut control. LdMNPV efficacy was unaffected by genetic transformation. Results suggest that although genetic modification of trees may affect interactions with other nontarget organisms, this may be due to insertion effects, and variation among different genotypes (whether transgenic or wild-type) imparts a greater change in response than transgene presence.

Characterization and functional assay of apsup (Lyxy105) from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV)

The baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation of viral infection. Recently, a new anti-apoptotic gene (apoptosis suppressor, apsup) was identified in Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). An apsup homolog gene, Lyxy105 (ly-apsup), was also predicted in the Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) genome. In this study, we attempt to perform a gene expression analysis and a functional assay of ly-apsup to demonstrate its anti-apoptotic activity and identify the functional domain of this protein. The transcription of the ly-apsup gene region was detected from 12h post-infection (hpi) and increased significantly after 24-72hpi. Comparison of the putative amino acid sequences to those of 18 baculoviral homolog proteins showed high amino acid identity to the LdMNPV sequences. Moreover, five conserved protein domains (named as domains I-V) were found. Therefore, protein functional assays were conducted on full-length proteins and different truncation clones. The overexpression of each clone was confirmed by western blot analysis, and the data revealed that a cleavage of ~ 5kDa at the N-terminal region of the full-length, domains I-IV (1-241) and I-III (1-178), proteins occurred. The results of the functional analysis showed that full-length Ly-apsup and Ly-apsup with domain I (1-70) could inhibit Drosophila-RPR protein (D-RPR)-induced and actinomycin D (ActD)-induced apoptoses. In addition, the domains I and I-II (1-126) regions showed higher anti-apoptotic activity than the other domains in both D-RPR-induced and ActD-induced cell apoptoses. In conclusion, domain I of Ly-apsup may play an important role in the anti-apoptotic activity of this protein; cleavage of the Ly-apsup N-terminus may lead to decreased anti-apoptotic activity.

Molecular Alliance of Lymantria dispar Multiple Nucleopolyhedrovirus and a Short Unmodified Antisense Oligonucleotide of Its Anti-Apoptotic IAP-3 Gene: A Novel Approach for Gypsy Moth Control.

Baculovirus IAP (inhibitor-of-apoptosis) genes originated by capture of host genes. Unmodified short antisense DNA oligonucleotides (oligoDNAs) from baculovirus IAP genes can down-regulate specific gene expression profiles in both baculovirus-free and baculovirus-infected insects. In this study, gypsy moth (Lymantria dispar) larvae infected with multiple nucleopolyhedrovirus (LdMNPV), and LdMNPV-free larvae, were treated with oligoDNA antisense to the RING (really interesting new gene) domain of the LdMNPV IAP-3 gene. The results with respect to insect mortality, biomass accumulation, histological studies, RT-PCR, and analysis of DNA apoptotic fragmentation suggest that oligoRING induced increased apoptotic processes in both LdMNPV-free and LdMNPV-infected insect cells, but were more pronounced in the latter. These data open up possibilities for promising new routes of insect pest control using antisense phosphodiester DNA oligonucleotides.

A comparison of the adaptations of strains of Lymantria dispar multiple nucleopolyhedrovirus to hosts from spatially isolated populations

The adaptation of pathogens to either their hosts or to environmental conditions is the focus of many current ecological studies. In this work we compared the ability of six spatially-distant Lymantria dispar (gypsy moth) multiple nucleopolyhedrovirus (LdMNPV) strains (three from eastern North America and three from central Asia) to induce acute infection in gypsy moth larvae. We also sequenced the complete genome of one Asian (LdMNPV-27/0) and one North American (LdMNPV-45/0) strain which were used for bioassay. We found that all of the North American virus strains, with the exception of one, demonstrated higher potency than the Asian virus strains, either in North American (Lymantria dispar) larvae or, in Asian (Lymantria dispar asiatica) larvae. Complete genome sequencing revealed two gene deletions in the LdMNPV-27/0 strain: the virus enhancin factor gene (vef-1) and the baculovirus repeated orf gene (bro-p). These deletions were not seen in the LdMNPV-45/0 strain nor in other American strains available in archiving systems. We also found deletions of the bro-e and bro-o genes in LdMNPV-45/0 strain but not in the LdMNPV-27/0 strain. The phylogenetic inference with an alignment of the 37 core gene nucleotide sequences revealed the close relationship of the LdMNPV-45/0 strain with other American strains accessed in GenBank (Ab-a624 and 5-6) while the LdMNPV-27/0 strain was clustered together with the LdMNPV-3054 strain (isolated in Spain) instead of predicted clustering with LdMNPV- 3029 (isolated in Asia). Our study demonstrated that first, different LdMNPV isolates from the same metapopulations of L. dispar exhibit little or no difference in the degree of virulence towards host larvae and second, that locality of host population is not an important driver of LdMNPV virulence. Virulence of LdMNPV is determined only by viral genetics. The genetic differences between North American and Central Asian virus strains are discussed.

Complete Genome Sequence of Lymantria dispar multiple nucleopolyhedrovirus Isolated in Southwestern Poland.

We report here the complete genomic sequence of Polish alphabaculovirus isolated from dead gypsy moth caterpillars. Its genome structure and G+C content indicate that it is a Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) strain which possesses the shortest sequence among eight other (similar to reference sequence AF081810) LdMNPV sequences available in GenBank; it is 159,729 bp long.

Baculovirus-induced tree-top disease: how extended is the role of egt as a gene for the extended phenotype?

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantriadispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L.dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographacalifornica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusiani and Spodopteraexigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T.ni and S.exigua larvae, although in S.exigua a moulting-dependent effect was seen. Those S.exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T.ni and S.exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a 'gene for an extended phenotype' is therefore not generally applicable for all baculovirus-host interactions. We hypothesize that in some baculovirus-host systems (including LdMNPV in L.dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour.

Classification, genetic variation and pathogenicity of Lymantria dispar nucleopolyhedrovirus isolates from Asia, Europe, and North America

Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) has been formulated and applied to control outbreaks of the gypsy moth, L. dispar. To classify and determine the degree of genetic variation among isolates of L. dispar NPVs from different parts of the range of the gypsy moth, partial sequences of the lef-8, lef-9, and polh genes were determined for Lymantria spp. virus samples from host populations throughout the world. Sequence analysis confirmed that all L. dispar virus samples tested contained isolates of the species Lymantria dispar multiple nucleopolyhedrovirus (Baculoviridae: Alphabaculovirus). Phylogenetic inference based on the lef-8 sequences indicated that the LdMNPV isolates formed two groups, one consisting primarily of isolates from Asia, and one consisting primarily of isolates from Europe and North America. The complete genome sequence was determined for an isolate from the Asian group, LdMNPV-2161 (S. Korea). The LdMNPV-2161 genome was 163,138bp in length, 2092bp larger than the previously determined genome of LdMNPV isolate 5–6 (CT, USA). The two genome sequences were co-linear, with an overall nucleotide sequence identity of 97.5% and some differences in ORF content. In droplet-feeding bioassays against neonate L. dispar larvae, isolates LdMNPV-3029 (Virin-ENSh/Russia) and LdMNPV-Ab-a624 (MA, USA) killed neonate larvae with an LC50 values that were 1.8- to 3.2-fold lower than a sample of Gypchek® (CT, USA) and isolates LdMNPV-3041 (Japan) and LdMNPV-2161. This study expands our knowledge about genetic variation among LdMNPV isolates and provides novel information on the distinct groups in which these NPVs occur.

Novel Apoptosis Suppressor Apsup from the Baculovirus Lymantria dispar Multiple Nucleopolyhedrovirus Precludes Apoptosis by Preventing Proteolytic Processing of Initiator Caspase Dronc

We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells.

Baculovirus Lymantria dispar multiple nucleopolyhedrovirus IAP2 and IAP3 do not suppress apoptosis, but trigger apoptosis of insect cells in a transient expression assay

Ld652Y cells derived from the gypsy moth, Lymantria dispar, are permissive for productive infection with L. dispar multiple nucleopolyhedrovirus (LdMNPV), but undergo apoptosis upon infection with various other NPVs, including those isolated from Bombyx mori, Hyphantria cunea, Spodoptera exigua, Orgyia pseudotsugata, and Spodoptera litura. In this study, we examined whether LdMNPV-encoded inhibitor of apoptosis 2 (Ld-IAP2) and 3 (Ld-IAP3) are involved in apoptosis suppression in LdMNPV-infected Ld652Y cells. We found that neither Ld-IAP2 nor Ld-IAP3 was able to suppress the apoptosis of Ld652Y cells induced by p35-defective Autographa californica MNPV (vAcΔp35). However, both Ld-IAP2 and Ld-IAP3 induced apoptosis in Ld652Y cells in a transient expression assay. The apoptosis induced by Ld-IAP3 was accompanied by the stimulation of caspase-3-like protease activity and cleavage of the B. mori homolog of the initiator caspase Dronc, and was precluded by the LdMNPV-encoded apoptosis suppressor protein Apsup and H. cunea MNPV IAP3. Inconsistent with the results obtained previously in SpIm, Ld652Y and High Five cells infected with NPVs from H. cunea, O. pseudotsugata, and A. californica, respectively, considerable stimulation of caspase-3-like protease activity was not observed in LdMNPV-infected Ld652Y cells, likely due to the strong apoptosis suppression activity of Apsup. These results, together with the previous finding that RNAi-mediated silencing of apsup induces apoptosis of LdMNPV-infected Ld652Y cells, indicate that Apsup, but not Ld-IAP2 or Ld-IAP3, is primarily responsible for the suppression of apoptosis in LdMNPV-infected Ld652Y cells. However, it remains inconclusive whether Ld-IAP2 and Ld-IAP3 function as pro-apoptotic proteins in LdMNPV-infected Ld652Y cells.

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD 50 in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48–72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.

Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.

Pathogenesis of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar and mechanisms of developmental resistance

Lymantria dispar has a long historical association with the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), which is one of the primary population regulators of L. dispar in the field. However, host larvae exhibit strong developmental resistance to fatal infection by LdMNPV; the LD50 in newly moulted fourth instars is 18-fold lower than in the middle of the instar (48-72 h post-moult). Using a recombinant of LdMNPV expressing lacZ, we examined the key steps of pathogenesis in the host to explore mechanisms of developmental resistance. At the midgut level, we observed reduced primary midgut infections in mid-fourth instars, indicating increased sloughing of infected cells. Additional barriers were observed as the virus escaped the midgut. Mid-fourth instars had higher numbers of melanized foci of infection associated with the midgut, apoptotic tracheal epidermal cells and haemocytes, and reduced numbers of infected haemocytes later in infection. Our results show that the co-evolutionary relationship between L. dispar and LdMNPV has resulted in both midgut-based and systemic antiviral defences and that these defences are age-dependent within the instar. This age-related susceptibility may contribute to how the virus is maintained in nature and could influence management of L. dispar by using the virus.

Functional analysis of the baculovirus host range gene, hrf-1

The hrf-1 gene from Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) prevents translation arrest and promotes Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication in IPLB-Ld652Y cells (Ld652Y), a non-permissive L. dispar cell line. There are no motifs in the predicted protein sequence to suggest how it might function and the only homolog identified is encoded by another baculovirus, Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). In this study, we report a functional analysis of the hrf-1 protein. AcMNPV bearing carboxy- or amino-terminally truncation hrf-1, and hrf-1 mutated by two-amino acid insertions did not replicate Ld652Y cells. Neither OpMNPV hrf-1 nor an OpMNPV/LdMNPV chimeric hrf-1 supported AcMNPV replication. Mutations in a highly acidic domain of hrf-1, in which aspartic acid residues were replaced with alanine, had varied effects on hrf-1 function. They had no effect, abolished hrf-1 function completely, or partially supported protein synthesis in infected Ld652Y cells. A slight increase in protein synthesis was achieved by increasing the expression of hrf-1 acidic domain mutant proteins. Together, these results indicate a critical role for hrf-1 structure and suggest a functional role for the acidic domain.