Prevalence of a Novel Bunyavirus in Tea Tussock Moth Euproctis pseudoconspersa (Lepidoptera: Lymantriidae).

Euproctis pseudoconspersa is a major pest of tea plants, and also causes a skin rash on workers in tea plantations. Research on virus could provide fundamental insights for classification, genetic diversity, evolution, and host-virus interaction mechanisms. Here, we identified a novel RNA virus, Euproctis pseudoconspersa bunyavirus (Phenuiviridae), and found that it is widely distributed in field populations of E. pseudoconspersa. The replication of virus in E. pseudoconspersa was indicated by Tag-PCR. These results contribute to the classification of bunyaviruses and provide insight into the diversity of commensal E. pseudoconspersa bunyavirus and the host.

Genomic analyses of Biston suppressaria nucleopolyhedrovirus: a viral isolate obtained from the tea looper caterpillar, Biston suppressaria (Guenée, 1857).

We described the complete genome sequence of a novel baculovirus isolate of species Buzura suppressaria nucleopolyhedrovirus, called by isolate CNPSo-25. The occlusion bodies were found to be polyhedral in shape and to contain virions with singly embedded nucleocapsids. The size of the genome is 121,377 bp with a G+C content of 36.7%. We annotated 131 ORFs that cover 90.42% of the genome. Moreover, phylogenetic inference indicated that CNPSo-25 is a member of genus Alphabaculovirus that clustered together with two other Chinese isolates of the same species. We called the virus by Biston suppressaria nucleopolyhedrovirus isolate CNPSo-25 (BisuNPV-CNPSo-25), as Buzura was placed inside the lepidopteran genus Biston. As expected, we detected intra-population variability in the virus sample when the novel isolate was compared to the Chinese isolates: 292 single nucleotide variants were found in the genome, with 181 affecting the protein product. The closest representatives of other species to BisuNPV-CNPSo-25 was found to be Sucra jujuba nucleopolyhedrovirus and Hyposidra talaca nucleopolyhedrovirus, two other virus isolates of geometrid caterpillars. The study of baculovirus genomes is of importance for the development of tools for insect pest biological control and biotechnology.

Development of a viral biopesticide for the control of the Guatemala potato tuber moth Tecia solanivora.

The Guatemala potato tuber moth Tecia solanivora (Povolny) (Lep. Gelechiidae) is an invasive species from Mesoamerica that has considerably extended its distribution area in recent decades. While this species is considered to be a major potato pest in Venezuela, Colombia, and Ecuador, currently no specific control methods are available for farmers. To address this issue we developed a biopesticide formulation to be used in integrated pest management of T. solanivora, following three steps. First, search for entomopathogenic viruses were carried out through extensive bioprospections in 12 countries worldwide. As a result, new Phthorimaea operculella granulovirus (PhopGV) isolates were found in T. solanivora and five other gelechid species. Second, twenty PhopGV isolates, including both previously known and newly found isolates, were genetically and/or biologically characterized in order to choose the best candidate for a biopesticide formulation. Sequence data were obtained for the ecdysteroid UDP-glucosyltransferase (egt) gene, a single copy gene known to play a role in pathogenicity. Three different sizes (1086, 1305 and 1353 bp) of egt were found among the virus isolates analyzed. Unexpectedly, no obvious correlation between egt size and pathogenicity was found. Bioassays on T. solanivora neonates showed a maximum of a 14-fold difference in pathogenicity among the eight PhopGV isolates tested. The most pathogenic PhopGV isolate, JLZ9f, had a medium lethal concentration (LC(50)) of 10 viral occlusion bodies per square mm of consumed tuber skin. Third, we tested biopesticide dust formulations by mixing a dry carrier (calcium carbonate) with different adjuvants (magnesium chloride or an optical brightener or soya lecithin) and different specific amounts of JLZ9f. During laboratory experiments, satisfactory control of the pest (>98% larva mortality compared to untreated control) was achieved with a formulation containing 10 macerated JLZ9f-dead T. solanivora larvae per kg of calcium carbonate mixed with 50 mL/kg of soya lecithin. The final product provides an interesting alternative to chemical pesticides for Andean farmers affected by this potato pest.

Histopathology and effect on development of the PhopGV on larvae of the potato tubermoth, Phthorimaea operculella (Lepidoptera: Gelechiidae).

Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4×10(8) granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 degrees C at pH 6.0 in the presence of 3 mM MgCl(2). Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.

Interactions among insect-resistant soybean genotypes extracts with populations of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) susceptible and resistant to its nucleopolyhedrovirus

Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is being used in Brazil as a biological insecticide. Host plant resistance of soybean to insects is been searched for and some authors have mentioned the interference of plant chemistry in virus efficiency. Interactions among soybean extracts of genotypes used as a source of resistance (PI 274454 and PI 227687) with different AgMNPV concentrations in populations of A. geatalis susceptible (S) and resistant (R) to the virus were studied at laboratory condition. Higher mortality was observed when larvae fed on diets with extracts of the soybean genotypes compared with those fed on a plain diet (control). The mean lethal concentration (LC50) was reduced about 10 ties in the S-population fed on diets containing PI 274454 extracts and different concentrations of AgMNPV, compared to control diet. Additive effect was predominantly observed when larvae fed on diets with extracts of soybean genotypes (PI 274454 and PI 227687) and AgMNPV for both larval populations. The pupal weight was negatively influenced by the extracts incorporated to the diets compared to control, for both larval populations, notably for R-population. The results suggest that, in general, leaf extracts of soybean resistant genotype did not cause any harmful effect on virus efficiency.

O nucleopoliedrovirus de Anticarsia gemmatalis (AgMNPV) tem sido utilizado como um inseticida biológico no Brasil. A resistência de plantas de soja a insetos tem sido pesquisada e alguns autores têm mencionado a interferência de substâncias químicas de plantas sobre a eficiência de vírus. As interações entre extratos de genótipos de soja utilizados como fontes de resistência (PI 274454 e PI 227687) com diferentes concentrações do AgMNPV em populações de A. gemmatalis suscetível (S) e resistente (R) ao vírus foram estudadas em condições de laboratório. Mortalidades elevadas foram observadas quando as larvas foram alimentadas com dietas contendo extratos dos genótipos de soja, em relação às larvas alimentadas com dieta artificial sem a presença de extratos (testemunha). A concentração letal média (CL50) foi reduzida em aproximadamente 10 vezes, na população s alimentada com dieta contendo extratos da PI 274454 e diferentes concentrações do AgMNPV, comparada à dieta testemunha. Um efeito aditivo foi predominantemente observado quando as larvas se alimentaram em dietas com extratos dos genótipos de soja (PI 274454 e PI 227687) e o AgMNPV, para ambas as populações (S e R). O peso de pupa foi negativamente influenciado pela dieta contendo os extratos em relação à dieta testemunha, para ambas as populações, com destaque para a população R. Os resultados indicam que, no geral, os extratos de folhas de genótipos de soja resistentes não causam efeitos negativos na eficiência do vírus.

Interactions among insect-resistant soybean genotypes extracts with populations of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) susceptible and resistant to its nucleopolyhedrovirus.

Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is being used in Brazil as a biological insecticide. Host plant resistance of soybean to insects is been searched for and some authors have mentioned the interference of plant chemistry in virus efficiency. Interactions among soybean extracts of genotypes used as a source of resistance (PI 274454 and PI 227687) with different AgMNPV concentrations in populations of A. geatalis susceptible (S) and resistant (R) to the virus were studied at laboratory condition. Higher mortality was observed when larvae fed on diets with extracts of the soybean genotypes compared with those fed on a plain diet (control). The mean lethal concentration (LC50) was reduced about 10 ties in the S-population fed on diets containing PI 274454 extracts and different concentrations of AgMNPV, compared to control diet. Additive effect was predominantly observed when larvae fed on diets with extracts of soybean genotypes (PI 274454 and PI 227687) and AgMNPV for both larval populations. The pupal weight was negatively influenced by the extracts incorporated to the diets compared to control, for both larval populations, notably for R-population. The results suggest that, in general, leaf extracts of soybean resistant genotype did not cause any harmful effect on virus efficiency.

Characterization of a Baculovirus newly isolated from the tea slug moth, Iragoidae fasciata.

The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC(50)) and lethal time (LT(50)) were conducted to test the susceptibility of I. fasciata larvae to the virus.

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 LC(50) value of 4.46 x 10(4) OBs/ml for the second instar caterpillars. Median lethal time (LT(50)) were 6.6 days for 1 x 10(4)OBs/ml, 5.09 days for 1 x 10(5) OBs/ml, 4.45 days for 1 x 10(6) OBs/ml and 3.87 days for 1 x 10(7) OBs/ml concentrations. The results indicated the potential of the virus for its future application as microbial pesticide against A. submarginata in future.

Evaluation of a granulovirus (PoGV) and Bacillus thuringiensis subsp. kurstaki for control of the potato tuberworm (Lepidoptera: Gelechiidae) in stored tubers.

Liquid suspensions and dry formulations of a granulovirus (family Baculoviridae, genus Granulovirus, PoGV) derived from infected larvae and the bacterium Bacillus thuringiensis subsp. kurstaki (Berliner) (Btk) were evaluated for control of the potato tuberworm, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), in stored tubers. Laboratory bioassays at 25 degrees C showed that both PoGV and a wettable powder (WP) formulation of Btk incorporated with carriers (water, talc, sand, diatomaceous earth, and kaolin clay), were effective against neonate larvae. Depending on the technique, 100% larval mortality was achieved at concentrations as low as 0.025 larval equivalents (LE) PoGV per kg tuber and 150 mg Btk WP per kg tuber. However, 100% mortality was never achieved with tests on preinfested tubers, ostensibly due to the higher dosage required to kill older instars inside tubers. The most effective PoGV formulations were dipping (water) and talc, with dipping most effective for postinfestation treatments, causing up to 91.6% mortality at 0.4 LE per kg. There was no significant effect of formulation in the Btk treatments. The protective effects of residues were also evaluated under longer-term storage conditions. Batches of tubers treated with PoGV or Btk via dipping (up to 0.1 LE and 150 mg WP per kg tuber) were stored in cages containing an initial potato tuberworm infestation (10% of tubers). Although potato tuberworm populations were reduced by up to 98.4% after 2 mo at 25 degrees C, no treatments prevented the development and reproduction of the F1 generation. The sprouting of stored tubers seemed to be a limiting factor for sustained control. No significant treatment effects were detected in similar cages held at 12 degrees C for 4.5 mo. Improved strategies for the application of PoGV and Btk for long-term potato tuberworm control in tuber stores, including the use of chemical sprout suppressants, are discussed.

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.

Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation.

Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.

Characterization of a newly established potato tuber moth (Phthorimaea operculella Zeller) cell line.

BACKGROUND & OBJECTIVE: Potato tuber moth (PTM), Phthorimaea operculella Zeller is a widely distributed, devastating pest of potatoes attacking the foliage and infest the tubers in both field and store causing serious economic damage. As application of PTM granulovirus (PTM-GV) has shown significant reduction in damage, attempts were made to develop a new cell line from this insect to grow PTM-GV for use as a biopesticide. METHODS: Approximately 100 mg of insect eggs were collected, surface sterilized and crushed gently in a boiling tube aseptically. The tissues were washed with physiological saline, suspended in growth medium and incubated stationary at 28 degrees C. Morphology of cells was studied after staining with Giemsa. Besides karyological and growth curve studies, PCR amplification was also done for rapid amplified polymorphic DNA pattern. RESULTS: A new cell line from the embryonic tissue of PTM was maintained in Mitsuhashi Maramorosch medium supplemented with 10 per cent foetal bovine serum. It is in the 78th passage level and designated as NIV-PTM-1095. Random amplified polymorphic DNA profile analysis indicated this as a new cell line from potato tuber moth and differed from the profiles of two other lepidopteran cell lines maintained in the laboratory. Three different cell types were observed at the 40th passage level and comprised of epithelial-like cells (77%), fibroblast-like cells (20%) and giant cells (3%). The chromosome number varied from 54-176. The cell line had a cell doubling time of approximately 42 h during the logarithmic phase of growth. The cell line did not support the multiplication of any of the baculoviruses used in the study. INTERPRETATION & CONCLUSION: Since the new cell line is found to replicate PTM-GV, it may be useful for the propagation of PTM-GV in large scale. Studies to scale up the production of the GV in the cell line and field trials may lead to its widespread use as an eco-friendly biopesticide.

Differential requirements of two insect cell lines for growth in serum-free medium.

The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.

Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain.

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response of P. includens toward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed in P. includens hemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0- and 1.5-kb cDNA clones (MdPi455 and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5' ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense against M. demolitor and other parasites.