Helicoverpa Zea Single Nucleopolyhedrovirus Research Articles

Host permissiveness to baculovirus influences time-dependent immune responses and fitness costs.

Insects possess specific immune responses to protect themselves from different types of pathogens. Activation of immune cascades can inflict significant developmental costs on the surviving host. To characterize infection kinetics in a surviving host that experiences baculovirus inoculation, it is crucial to determine the timing of immune responses. Here, we investigated time-dependent immune responses and developmental costs elicited by inoculations from each of two wild-type baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa zea single nucleopolyhedrovirus (HzSNPV), in their common host H. zea. As H. zea is a semi-permissive host of AcMNPV and fully permissive to HzSNPV, we hypothesized there are differential immune responses and fitness costs associated with resisting infection by each virus species. Newly molted 4th-instar larvae that were inoculated with a low dose (LD15 ) of either virus showed significantly higher hemolymph FAD-glucose dehydrogenase (GLD) activities compared to the corresponding control larvae. Hemolymph phenoloxidase (PO) activity, protein concentration and total hemocyte numbers were not increased, but instead were lower than in control larvae at some time points post-inoculation. Larvae that survived either virus inoculation exhibited reduced pupal weight; survivors inoculated with AcMNPV grew slower than the control larvae, while survivors of HzSNPV pupated earlier than control larvae. Our results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.

CHARACTERIZATION OF HELICOVERPA ZEA SINGLE NUCLEOPOLYHEDROVIRUS ISOLATED IN BRAZIL DURING THE FIRST OLD WORLD BOLLWORM (NOCTUIDAE: HELICOVERPA ARMIGERA) NATIONWIDE OUTBREAK

A baculovirus isolated in Brazil during the first nationwide outbreak of Helicoverpa armigerais described by ultrastructural analyses, restriction profiles, pathogenicity of host insects, and complete genome sequence. The results revealed that the virus is an isolate of the species Helicoverpa zea single nucleopolyhedrovirus(HzSNPV-Brazilian) never reported before in Brazil. Among the HzSNPV isolates few mutations were observed depicting likely a recent divergence of this lineage. Therefore, the entrance of both foreign pests and natural pathogens into the country must warn the government to reinforce sanitary barriers in order to avoid possible agriculture sabotage and novel foreign pest introductions. Moreover, we found that the Brazilian natural isolate was as lethal as a commercial strain to H. armigera.Importantly, virus characterization is of importance in establishment of an economical and useful virusbased biological control program in the country to counteract effectively pest infestations. DOI: http://dx.doi.org/10.17525/vrrjournal.v20i1.254

Gut Transcription in Helicoverpa zea is Dynamically Altered in Response to Baculovirus Infection.

The Helicoverpa zea transcriptome was analyzed 24 h after H. zea larvae fed on artificial diet laced with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Significant differential regulation of 1,139 putative genes (p < 0.05 T-test with Benjamini and Hochberg False Discovery Rate) was detected in the gut epithelial tissue; where 63% of these genes were down-regulated and 37% of genes were up-regulated compared to the mock-infected control. Genes that play important roles in digestive physiology were noted as being generally down-regulated. Among these were aminopeptidases, trypsin-like serine proteases, lipases, esterases and serine proteases. Genes related to the immune response reacted in a complex nature having peptidoglycan binding and viral antigen recognition proteins and antiviral pathway systems down-regulated, whereas antimicrobial peptides and prophenoloxidase were up-regulated. In general, detoxification genes, specifically cytochrome P450 and glutathione S-transferase were down-regulated as a result of infection. This report offers the first comparative transcriptomic study of H. zea compared to HzSNPV infected H. zea and provides further groundwork that will lead to a larger understanding of transcriptional perturbations associated with viral infection and the host response to the viral insult in what is likely the most heavily infected tissue in the insect.

Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro

A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infected with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) accumulated hsp70 transcripts throughout the 72-hour course of infection in the midgut, hemocytes, and fat body. While a maximal 17- or 15-fold induction of hsp70 was noted in the midgut and hemocytes, respectively, by 72hours postinfection, the level of hsp70 transcription in the fat body of larvae was greater than two orders of magnitude higher than in mock-infected larvae. These results were largely mirrored in cultures of infected cells, and a potentiation effect was observed in cells that were both heat shocked and infected. In contrast, Spodoptera frugiperda multiple nucleopolyhedrovirus and ultraviolet-inactivated HzSNPV did not stimulate hsp70 transcription in these non-permissive larvae and in cell culture, respectively. Taken together, this report documents baculovirus-mediated upregulation of hsp70 in the host and demonstrates the requirement for productive infection for hsp70 induction in vitro and in vivo.

Iron Levels Change in Larval Heliothis virescens Tissues Following Baculovirus Infection

Inductively coupled plasma mass spectrometry and (59)Fe radiotracers were used to investigate changes in levels of Fe in the tissues of 4th instar Heliothis virescens larvae following infection with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) or with Autographa californica multiple nucleopolyhedrovirus. Baculovirus infection led to significant changes in hemolymph Fe levels late in infection. (24)Na radiotracer ingested by 4th instar larvae was rapidly cleared to nearly undetectable levels 6 h post-ingestion. In contrast, (59)Fe radiotracer fed to 4th instar larvae declined within the first few hours of ingestion and then remained constant at approximately 60% of the initial tracer activity. While Fe radiotracer levels among larval tissues changed, whole insect tracer levels did not decline from 6 to 60 h post-ingestion. Tissues from HzSNPV larvae had higher radiotracer levels in the hemolymph and midgut 36 and 60 h post-infection. The protein-bound/free ratio of (59)Fe was significantly higher in baculovirus infected hemolymph than in uninfected hemolymph at 60 h post-infection, indicating that Fe released from damaged cells is protein-bound. In both studies, hemolymph Fe levels were higher in HzSNPV infected larvae. This first study of tissue Fe levels during viral infection of an insect clearly demonstrates that Fe homeostasis is substantially disrupted.

Baculovirus Induced Transcripts in Hemocytes from the Larvae of Heliothis virescens

Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae.

Analysis of ESTs generated from immune-stimulated hemocytes of larval Heliothis virescens

Heliothis virescens immunome components responding to baculoviral and bacterial infection were identified from expressed sequence tags (ESTs) generated from an immune-stimulated larval hemocyte cDNA library. A total of 5548 ESTs were generated comprising 448 contigs and 1114 singletons, totaling 1606 putative transcripts 1101 of which had BLAST scores, including many known orthologs from other insect species. Orthologs of known or putative immune function were identified among them melanization pathway components, proteases, antibacterial proteins, lectins, bacteria-binding proteins, ferritins, scavenger receptors, cell surface receptors, signaling pathway components, and stress response enzymes. Additionally, many enzymes of central metabolism, cytoskeletal, mitochondrial, and ribosomal components, as well as transcriptional and translational regulators were identified. The effect of bacterial and baculoviral infection upon transcript levels of three identified immunome targets from among the ESTs was quantitated using real time PCR. Scolexin-B, C-type lectin and growth-blocking peptide binding protein transcripts were significantly elevated by bacterial infection. Per os infection with the baculovirus Helicoverpa zea single nucleopolyhedrovirus however did not significantly alter transcript levels of these three genes. The ESTs reported here are the first large scale report of the H. virescens immunome responding to entomopathogens, and represent a first step to a more complete transcriptome for this pest moth.

Increased plasma selenium levels correlate with elevated resistance of Heliothis virescens larvae against baculovirus infection

We reported that dietary selenium (Se) impacted the growth and development of Trichoplusia ni reared for many generations on diet containing extremely low levels of Se. Larvae had an elevated resistance to per os infection with a baculovirus. In this study, we examine how dietary Se (in the form of selenite) affects the growth, development, and Se content of Heliothis virescens that have been laboratory reared for less than two years. Larvae fed a commercial tobacco budworm diet supplemented with greater than 20 ppm Se grew at a slower rate than insects fed lower levels of Se and had an increase in the amount of Se sequestered in pupae. Larvae fed diets containing from 10–60 ppm Se exhibited elevated plasma concentrations of the micronutrient and increased plasma virucidal activity against Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Larvae reared on diet supplemented with 10 or 60 ppm Se until the onset of the penultimate instar were then infected per os or by injection with increasing concentrations of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed dietary Se and infected with occluded virus per os displayed a significantly lower mortality compared with infected larvae not fed Se. Our results suggest that dietary Se levels are directly correlated with plasma Se levels, and that plasma Se levels are in turn correlated with baculovirus resistance.

Plasma phenoloxidase of the larval tobacco budworm,Heliothis virescens,is virucidal

Heliothis virescens larval plasma contains high levels of an antiviral activity against the budded form of the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV) in vitro. Preliminary results indicated that phenoloxidase is primarily responsible for this virucidal effect. However it is known that other enzymes that generate antimicrobial reactive oxygen intermediates and reactive nitrogen intermediates are present in hemolymph that could contribute to the observed virucidal activity. To elucidate the contributions of phenoloxidase and other candidate activities to plasma innate immune response against baculovirus infection specific metabolic inhibitors were used. In vitro the general inhibitors of melanization (N-acetyl cysteine, ascorbate and glutathione), and specific inhibitors of phenoloxidase (phenylthiourea and Kojic acid), completely blocked virucidal activity up to the level seen in controls. Addition of the enzyme superoxide dismutase to plasma did not affect virucidal activity; however addition of catalase had an inhibitory effect. Inhibitors of nitric oxide synthase activity did not affect virucidal activity. Our results confirm that phenoloxidase is the predominate activity in larval plasma accounting for inactivation of Hz SNPV in vitro, and that phenoloxidase-dependent H2O2 production may contribute to this virucidal activity.

Insect cells and their potential as stabilization barriers for DNA of multiple and single nucleopolyhedroviruses against ultraviolet-B-simulated sunlight inactivation.

A cell line from Trichoplusia ni (TN-CL1) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.

Molecular characterization of genes in the GP41 region of baculoviruses and phylogenetic analysis based upon GP41 and polyhedrin genes

A newly sequenced Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) gp41 gene was used to reconstruct the phylogeny for gp41 by comparison with Autographa californica MNPV, Bombyx mori MNPV, Helicoverpa zea single nucleopolyhedrovirus (SNPV), Lymantria dispar MNPV, Orgyia pseudotsugata MNPV and Spodoptera frugiperda MNPV. The 3.5 kb fragment of the AgMNPV gp41 region not only contained the gp41 gene but also three other open reading frames that had significant homology with the very late factor (vlf-1) of baculoviruses, AcMNPV ORF78, AcMNPV ORF79, and one partial open reading frame homologous to AcMNPV ORF81. The reconstructed phylogenetic tree of baculovirus gp41 genes compared with the polyhedrin gene tree produced similar topologies. Two other phylogenetic trees were reconstructed based on either combined gp41 and polyhedrin nucleotide sequences (total evidence) or combined evolutionary histories of both genes (strict consensus tree). The former had an identical tree topology as the gp41 gene tree alone, and the latter lost resolution in the branch of AcMNPV and BmMNPV. Mutation rate analysis showed the gp41 gene had a higher nucleotide substitution rate than the polyhedrin gene, implying that the polyhedrin gene may have a different selection constraint than the gp41 gene. Both genes have nonsynonymous/synonymous substitution values close to 0.1, similar to other DNA viruses.