Nuclear Polyhedrovirus Research Articles

A Lipidome Map of the Silkworm Bombyx mori: Influences of Viral Infection.

Lipids have been recently proposed as key molecules for virus entry and egress, and lipid biosynthesis and signaling were reported necessary for some viruses during replication and infection. The silkworm Bombyx mori is an important economic insect and a model organism, but its lipid profiles have not been systematically investigated. Most silkworm strains are susceptible to the B. mori nuclear polyhedrovirus (BmNPV), a baculovirus that causes serious loss to the sericulture industry. Previously, our lab has screened a natural mutant of B. mori that is highly resistant to BmNPV. In this study, a comprehensive lipidomic analysis by ultrahigh pressure liquid chromatography-mass spectrometry (UPLC-MS) was carried out on the BmNPV-susceptible strain 306 and resistant strain NB (data deposited in MetaboLight MTBLS2142). Comparisons of the lipid profiles between the two strains reveal that phosphosphingolipids, diacylglycerolipids, ceramides, and quinones were present at notably higher levels in the susceptible strain, while lysophosphocholines were found at a higher level in the resistant strain. BmNPV administration changed the lipid profiles in both strains, revealing key lipids involved in virus infection and immune response. Some key enzymes in the lipid biosynthesis pathway were analyzed for their activities in the two silkworm strains and their virus-administered counterparts, underlining the relation among lipid biosynthesis, viral resistance, and immune response in the host.

Intraguild predation decreases predator fitness with potentially varying effects on pathogen transmission in a herbivore host

Predators and pathogens often regulate the population dynamics of their prey or hosts. When species interact with both their predators and their pathogens, understanding each interaction in isolation may not capture the system's dynamics. For instance, predators can influence pathogen transmission via consumptive effects, such as feeding on infected prey, or non-consumptive effects, such as changing the prey's susceptibility to infection. A prey species' infection status can, in turn, influence predator's choice of prey and have negative fitness consequences for the predator. To test how intraguild predation (IGP), when predator and pathogen share the same prey/host, affects pathogen transmission, predator preference, and predator fitness, we conducted a series of experiments using a crop pest (Pseudoplusia includens), a generalist predator (Podisus maculiventris), and a generalist pathogen (Autographa californica multicapsid nuclear polyhedrovirus, AcMNPV). Using a field experiment, we quantified the effects of consumptive and non-consumptive predators on pathogen transmission. We found that a number of models provided similar fits to the data. These models included null models showing no effects of predation and models that included a predation effect. We also found that predators consumed infected prey more often when choosing between live infected or live healthy prey. Infected prey also reduced predator fitness. Developmental times of predators fed infected prey increased by 20% and longevity decreased by 45%, compared with those that consumed an equivalent number of non-infected prey. While this research shows an effect of the pathogen on intraguild predator fitness, we found no support that predators affected pathogen transmission.

Bombyx mori Rad23 (BmRad23) contributes to the repair of UV-damaged BmNPV

Baculoviruses have been developed as long-term and environmentally friendly biopesticides. However, solar ultraviolet radiation can reduce the activity of baculovirus. Radiation sensitive 23 (Rad23) can recognize DNA damage and is involved with nucleotide excision repair (NER). In the current study, BmRad23 was accumulated mainly within the nucleus. Host cell reactivation (HCR) assays have shown that BmRad23 significantly facilitated the expression of UV-damaged mCherry reporter gene. Reverse transcription quantitative PCR (RT-qPCR) result showed that the mRNA expression level of BmRad23 was increased in (Bombyx mori nuclear polyhedrovirus, BmNPV) BmNPV-infected BmN cells. However, the expression of BmRad23 was increased significantly when BmNPV budded viruses (BVs) or BmN cells were irradiated with UV light. Overexpression of BmRad23 promoted the mRNA levels of two UV-induced DNA damage repair genes which were from Bombyx mori and BmNPV, respectively. Meanwhile, the overexpression of BmRad23 in BmN cells was conducive to the proliferation of BmNPV and UV-damaged BmNPV. The recombinant BmNPV BVs expressing BmRad23 showed stronger resistance to UV radiation than the control virus. In conclusion, the results revealed that BmRad23 contributed to the proliferation of BmNPV and the repair of UV-damaged BmNPV, which would provide a reference for the development of efficient baculovirus pesticides against UV radiation.

Expression and nuclear localization of the TATA-box-binding protein during baculovirus infection

The TATA-box-binding protein (TBP) plays a key role in initiating eukaryotic transcription and is used by many viruses for viral transcription. We previously reported increased TBP levels during infection with the baculovirus Autographa californica multicapsid nuclear polyhedrovirus (AcMNPV). The TBP antiserum used in that study, however, cross-reacted with a baculoviral protein. Here, we reported that increased amounts of nuclear TBP were detected upon infection of Spodoptera frugiperda and TN-368 cells with a TBP-specific antiserum. TBP levels increased until 72 h post-infection (p.i.), whilst tbp transcripts decreased by 16 h p.i., which suggested a virus-induced influence on the TBP protein levels. To address a potential modification of the TBP degradation pathway during infection, we investigated the possible role of viral ubiquitin. Infection studies with AcMNPV recombinants carrying a mutated viral ubiquitin gene revealed that the TBP increase during infection was not altered. In addition, pulse-chase experiments indicated a high TBP half-life of ~60 h in uninfected cells, suggesting that a virus-induced increase of TBP stability was unlikely. This increase in TBP correlated with a redistribution to nuclear domains resembling sites of viral DNA synthesis. Furthermore, we observed colocalization of TBP with host RNA polymerase (RNAP) II, but only until 8 h p.i., whilst TBP, but not RNAPII, was present in the enlarged replication domains late during infection. Thus, we suggested that AcMNPV adapted a mechanism to accumulate the highly stable cellular TBP at sites of viral DNA replication and transcription.

Protein Tyrosine Phosphatase-Induced Hyperactivity Is a Conserved Strategy of a Subset of BaculoViruses to Manipulate Lepidopteran Host Behavior

Many parasites manipulate host behavior to increase the probability of transmission. To date, direct evidence for parasitic genes underlying such behavioral manipulations is scarce. Here we show that the baculovirus Autographa californica nuclear polyhedrovirus (AcMNPV) induces hyperactive behavior in Spodoptera exigua larvae at three days after infection. Furthermore, we identify the viral protein tyrosine phosphatase (ptp) gene as a key player in the induction of hyperactivity in larvae, and show that mutating the catalytic site of the encoded phosphatase enzyme prevents this induced behavior. Phylogenetic inference points at a lepidopteran origin of the ptp gene and shows that this gene is well-conserved in a group of related baculoviruses. Our study suggests that ptp-induced behavioral manipulation is an evolutionarily conserved strategy of this group of baculoviruses to enhance virus transmission, and represents an example of the extended phenotype concept. Overall, these data provide a firm base for a deeper understanding of the mechanisms behind baculovirus-induced insect behavior.

Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori

A cell line BmN-SWU1 was established from the ovarian tissues of 3-day-old fourth instar Bombyx mori larvae of the 21-872nlw variety by performing primary cultures in Grace's medium supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of short spindle cells and round cells. The frequency of cells with chromosome number 2 n = 56 was 80.5%; therefore, the cell line was considered to be a diploid cell line. The population-doubling time (PDT) at 45th passage line was 57.7 h. This cell line was susceptible to the B. mori nuclear polyhedrovirus (BmNPV), and the median tissue culture infective dose (TCID 50) at a cell density of 10 5 cells/ml was 16.3 OBs/ml. The transient expression efficiency of the green fluorescent protein (GFP) gene in this cell line was 54.8%. We used the BmN-SWU1 cell line to select and establish a GFP transgenic cell line.

Identification and Functional Analysis of LsMNPV Anti-apoptosis Genes

Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD(74) downward arrow G) that is different from Sl-P49 (TVID(94) downward arrow G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assay revealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells.

P13 of Leucania separata multiple nuclear polyhedrosis virus affected the polyhedra and budded virions yields of AcMNPV

p13 gene was first described by our laboratory in Leucania separata multiple nuclear polyhedrovirus (Ls -p13, ORF114) back to 1995. However, the functions of Ls-P13 and its reported homologues remained unknown. In order to probe the function of Ls-P13, recombinant Autographa californica nucleopolyhedroviruses (rAcMNPVs) were constructed to express Ls-P13 in the Sf9 cells at early, late or early/late phase. Observations of microscope showed that the expression of Ls-P13 could decrease the yield of AcMNPV polyhedra in Sf9 cells, and early expressed Ls-P13 had stronger inhibition efficiency than that of the late expressed. Results of flow cytometry also indicated that Ls-P13 decreased the yield of AcMNPV polyhedra while increased those of budded virions (BVs) in Sf9 cells, but the efficacy was lost when its leucine zipper-like domain was mutated. Ls-P13 is a transmembrane protein, which was early located in the nucleus and late mainly in the cytoplasm membrane at 48 h. When its transmembrane domains were deleted, Ls-P13 distribution was dramatically diverted from cytoplasm membrane to nucleus, its corresponding efficacy on polyhedra yield was further increased while that on BVs was slightly weakened. Bioassay results indicated that Ls-P13 accelerated the larvae-killing rate. The mechanism might be that Ls-P13 increased BV yield.

Enzyme kinetics and glycan structural characterization of secreted alkaline phosphatase prepared using the baculovirus expression vector system.

Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that Vmax and Km of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the Vmax and Km of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man(2-8)GlcNAc2 and FucMan(3 or 4)GlcNAc2) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man4GlcNAc2 and FucMan4GlcNAc2 were the major rSEAP N-glycans produced in Sf-9 cells, while Man2GlcNAc2 was the major rSEAP N-glycan produced in Tn-5B1-4 cells.

Stability and Persistence of Two Formulations Containing Anticarsia gemmatalis Nuclear Polyhedrovirus (AgMNPV)

The stability and the persistence of two AgMNPV formulations developed by the Instituto Biológico and the Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Brazil were studied. Formulations were stored in environmental conditions and exposed to ultraviolet radiation in laboratory and to solar radiation in the field. The wettable powder formulation (WP) was prepared by impregnating viral suspensions + adjuvants on inert mineral, chiefly kaolin. The emulsifiable oil formulation (EO) was prepared by mixing the pathogen suspension with corn oil + adjuvants. At 120-day intervals for 20 months, samples of formulations and the standard treatment (crude virus) kept in a laboratory cabinet were added to artificial diet fed to 4th-instar Anticarsia gemmatalis (Hübner) larvae. After 20 months in storage, the formulation EO lost only 18.3% of its original activity, whereas the formulation WP had its efficiency reduced to 11.7% after 12 months. No significant differences between the two formulations were observed when exposed to the ultraviolet germicidal light, and both formulations protected the virus when compared to the standard treatment. Under field conditions, the EO formulation enhanced the viral persistence, with >60% of the original activity present 14 days after the application. Most of the WP formulation was removed from leaves by the rain.

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system.

Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.

Population Density and Transmission of Virus in Experimental Populations of the Western Tent Caterpillar (Lepidoptera: Lasiocampidae)

Epizootics of nuclearpolyhedrovirus (NPV) are associated with population fluctuations of western tent caterpillars, Malacosoma californicum pluviale (Dyar), in western Canada. To experimentally quantify the spread of NPV in the western tent caterpillar, we measured the transmission of NPV within and among colonies of larvae introduced to alder trees. We altered both host population size (number of larvae per colony on a tree with 4 colonies) and pathogen density (number of infected individuals introduced to 1 of 4 colonies per tree). At the scale of the tree, the transmission coefficient (proportion of new infections per initially infected caterpillar) was independent of host density, but varied with initial pathogen dose. Low doses of virus had higher transmission coefficients than high doses. Transmission of virus between colonies within trees was related to the size of the colonies and virus was more widely spread among the 4 colonies when colonies were large (256 larvae per colony). This could be attributed to variation in the development rate and mobility of caterpillars from small and large colonies. The dynamics of NPV in populations of tent caterpillars will be influenced by movement of caterpillars among colonies, but increases in the amount of virus above a threshold level will not lead directly to increased numbers of infected individuals. Individual tent caterpillar moths lay all of their eggs in a single egg mass, and in declining populations, egg masses are smaller. The slower development of small colonies arising from these smaller egg masses may delay the recovery of declining populations, restrict the spread of virus, and influence the population dynamics of tent caterpillars.