Identification of a new nucleopolyhedrovirus from naturally-infected Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae) larvae on poplar plantations in South Brazil

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81 ± 2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [³⁵S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.     

Characterization of a single-nucleocapsid nucleopolyhedrovirus of Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia

A single-nucleocapsid nucleopolyhedrovirus (NPV) isolated from Thysanoplusia orichalcea L. (Lepidoptera:Noctuidae) (ThorNPV) in Indonesia has tetrahedral occlusion bodies (OBs) with a width of 1. 22 microm (range = 0.803-1.931 microm). The length of the virion with an envelope averaged 0.29 and 0.23 microm without an envelope. ThorNPV was propagated in Pseudoplusia includens (Walker) and its authenticity was confirmed by sequence analysis of the polyhedrin gene of the ThorNPV produced in T. orichalcea and P. includens. Polyhedrin amino acid sequence analysis revealed that ThorNPV belongs to Group II of baculoviruses and is closely related to Trichoplusia ni single nucleocapsid NPV, sharing 97.6% sequence identity. Infectivity of ThorNPV against third instar P. includens was low, with a LD(50) value of 65,636 OBs/larva. Electron microscopy of infected tissues showed many polyhedra without virions embedded, which might explain the low virulence against P. includens. Differences in virion occlusion rates between individual cells in the same tissue suggested that the inoculum consisted of at least two variants that differed in the gene(s) controlling virion occlusion. In a host range test using the LD(50) value to P. includens against Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens, P. includens was the only species infected. The virus infected primarily the fat body, tracheal epithelium, and hypodermis. The genomic size of the ThorNPV is 135 kb.     

Characterization of a nucleopolyhedrovirus of Epinotia granitalis (Lepidoptera: Tortricidae)

A nucleopolyhedrovirus (NPV) from the cypress bark moth Epinotia granitalis Butler was partially characterized. Electron microscopy indicated that E. granitalis NPV (EpgrNPV) is a singly embedded nucleocapsid NPV with a relatively small polyhedron, ranging from 480 to 1100nm in diameter. Phylogenetic analysis revealed that EpgrNPV is a Group II NPV that is related to but distinct from Adoxophyes honmai NPV. The LC(50) against third instar E. granitalis larvae was 10(5.5) polyhedral occlusion bodies per gram (OBs/g diet), and the average time to death was 14 days. E. granitalis was the only EpgrNPV-susceptible species in a series of infection tests using insect species belonging to the family Tortricidae. Other test species, including Eucoenogenes ancyrota, A. honmai, Adoxophyes dubia, Pandemis heparana, Homona magnanima, and Archips insulanus, were not susceptible to the infection.     

Molecular characterization of Agrotis segetum nucleopolyhedrovirus from Poland

The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an isolate from England/France (AgseNPV-UK and AgseNPV-F). Sequence analysis of three conserved baculovirus genes, polyhedrin, lef-8 and pif-2, revealed that AgseNPV-P differs substantially from the already described NPVs isolated from A. segetum and possibly represents a new NPV species. Phylogenetic analysis placed AgseNPV-P among group II NPVs and showed the closest relationship to Agrotis ipsilon (Agip) NPV and Spodoptera exigua (Se) MNPV.     

Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

Susceptibility of mosquito and lepidopteran cell lines to the mosquito iridescent virus (IIV-3) from Aedes taeniorhynchus

Mosquito iridescent viruses (MIV) are members of the genus Chloriridovirus that currently contains only the type IIV-3 from Aedestaeniorhynchus. The complete genome of invertebrate iridescent virus -3 (IIV-3) has been sequenced and the availability of a tissue culture system would facilitate functional genomic studies. This investigation, using quantitative PCR and electron microscopy, has determined that the mosquito cell lines Aedes aegypti (Aag2), Aedes albopictus (C6/36) and Anopheles gambiae (4a3A) as well as the lepidopteran cell line from Spodoptera frugiperda (SF9) are permissive to IIV-3 infection. However, IIV-3 infection remained longer in Aag2 and C6/36 cells. Virus produced in C6/36 cell line was infectious to larvae of A. taeniorhynchus by injection and per os. Ultrastructural examination of 4a3A and SF9 cells infected with IIV-3 revealed an unusual feature, where virions were localized to mitochondria. It is speculated that containment with mitochondria may play a role in the lack of persistence in these cell lines.     

A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.     

Pathogenecity of a baculovirus isolated from Arctornis submarginata (Walker) (Lepidoptera:Lymantriidae), a potential pest of tea growing in the Darjeeling foothills of India

A granulosis virus (GV) was isolated from the diseased caterpillars of Arctornis submarginata (Walker) (Lymantriidae), a defoliating pest of tea from Darjeeling foothill region. The phase contrast and transmission electron microscopic studies identified the virus as granulosis virus. SDS-PAGE analysis of major protein of the occlusion bodies was found to be 31 kDa, characteristic for granulin. The total genomic DNA was isolated. The major band found was of molecular weight 16 kDa. Bioassay conducted with the occlusion bodies (OBs) of the virus showed LC₅₀ value of 4.46 × 10⁴ OBs/ml for the second instar caterpillars. Median lethal time (LT₅₀) were 6.6 days for 1 × 10⁴ OBs/ml, 5.09 days for 1 × 10⁵ OBs/ml, 4.45 days for 1 × 10⁶ OBs/ml and 3.87 days for 1 × 10⁷OBs/ml concentrations. The results indicated the potential of the virus for its future application as microbial pesticide against A. submarginata in future.     

A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.     

Differential requirements for baculovirus late expression factor genes in two cell lines.

A plasmid library of 18 late expression factor (LEF) genes (LEF library) from the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) supports transient expression from a late viral promoter in the SF-21 cell line, derived from Spodoptera frugiperda. We found, however, that this LEF library was unable to support expression from the same promoter in the TN-368 cell line, derived from Trichoplusia ni, which is also permissive for AcMNPV replication. To identify the additional factor(s) required for expression in TN-368 cells, we cotransfected the LEF library with clones representing portions of the AcMNPV genome not represented in the LEF library. A single additional gene was identified; this gene corresponded to ORF70 of the complete AcMNPV sequence and potentially encodes a 34-kDa cysteine-rich polypeptide. Because of its differential effect on late gene expression in the two cell lines, we renamed ORF70 hcf-1 (for host cell-specific factor 1). hcf-1 was involved in expression from reporter plasmids under late and very late but not early promoter control, indicating that it was also a LEF gene. Plasmid DNA replication assays indicated that HCF-1 was involved in virus origin-specific DNA replication in TN-368 cells. Three LEF genes, ie-2, lef-7, and p35, required for optimal virus origin-specific plasmid DNA replication or stability in SF-21 cells had little or no influence in TN-368 cells. Thus, as determined by transient-expression assays, cell line-specific and potentially host-specific factors are required for origin-specific DNA replication or stability.     

Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L

Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny.     

Characterisation of a Colombian granulovirus (Baculoviridae: Betabaculovirus) isolated from Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

A strategy for biological control of the fall armyworm, Spodoptera frugiperda, has included the use of baculoviruses principally the nucleopolyhedrovirus SfMNPV, which have been extensively characterised. In contrast, the granulovirus of S. frugiperda (SfGV) has been poorly studied even though it is able to enhance the infectivity and virulence of NPVs. In this work, a Colombian SfGV isolate (VG008) was characterised in comparison with a reference isolate from Brazil (VG014). The viral morphology was characterised by ovoidal-shaped occlusion bodies (OB) that contained one single internal virion. Median lethal concentrations (LC₅₀) and mean times to death (MTD) were 4.5 × 10⁵ OBs/mL and 29 days for VG008 and 1.6 × 10⁵ OBs/mL and 33 days for VG014. Both isolates reduced their insecticidal activity by 94%, after one hour of direct irradiation with ultraviolet light type B. The most prominent protein had an apparent molecular mass of 27 kDa and corresponded with the Granulin. Genomic comparison among isolates from Colombia and Brazil generated by restriction profiles showed differences in the number and size of fragments. Partial sequences of lef-8 and lef-9 genes and complete sequence of gran gene of Colombian SfGV isolate (VG008) showed high similarity values with VG014 and SfGV A12-4 homologous sequences, showing genetic distance lower than 0.015 (Kimura 2-parameter model), which confirmed that the three isolates belong to the same viral species. The characterisation of VG008 isolate demonstrated its high genomic and biological similarity with the Brazilian isolate.     

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells. However, occluded viruses expressing cv-PDG were not more resistant to UV inactivation than wild type AcMNPV when fed to either S. frugiperda or Trichoplusia ni neonate larvae. Surprisingly, however, viruses expressing cv-PDG showed a significant decrease in both the dose of occluded virus required for oral lethality and the time required for lethality compared to control virus, but these effects were only seen in S. frugiperda and not in T. ni larvae.     

Morphological, phylogenetic and biological characteristics of Ectropis obliqua single-nucleocapsid nucleopolyhedrovirus

The tea looper caterpillar, Ectropis obliqua, is one of the major pests of tea bushes. E. obliqua single-nucleocapsid nucleopolyhedrovirus (EcobSNPV) has been used as a commercial pesticide for biocontrol of this insect. However only limited genetic analysis for this important virus has been done up to now. EcobSNPV was characterized in this study. Electron microscopy analysis of the occlusion body showed polyhedra of 0.7 to 1.7 mum in diameter containing a single nucleocapsid per envelope of the virion. A 15.5 kb genomic fragment containing EcoRI-L, EcoRI-N and HindIII-F fragments, was sequenced. Analysis of the sequence revealed that the fragment contained eleven potential open reading frames (ORFs): lef-1, egt, 38.7k, rr1, polyhedrin, orf1629, pk-1, hoar and homologues to Spodoptera exigua multicapsid NPV (SeMNPV) ORFs 15, 28, and 29. Gene arrangement and phylogeny analysis suggest that EcobSNPV is closely related to the previously described Group II NPV. Bioassays on lethal concentration (LC(50) and LC(90)) and lethal time (LT(50) and LT(90)) were conducted to test the susceptibility of E. obliqua larvae to the virus.     

Establishment of a cell line from Chrysodeixis chalcites permissive for Chrysodeixis chalcites and Trichoplusia ni nucleopolyhedrovirus

A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.     

Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno

Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.     

Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae

It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVL-infected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ≤ vERTVL < vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae.