The \u3ci\u3eLymantria dispar\u3c/i\u3e Nucleopolyhedrovirus Enhancins Are Components of Occlusion-Derived Virus

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier

The \u3ci\u3eLymantria dispar\u3c/i\u3e Nucleopolyhedrovirus Enhancins Are Components of Occlusion-Derived Virus

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier

Both \u3ci\u3eLymantria dispar\u3c/i\u3e Nucleopolyhedrovirus \u3ci\u3eEnhancin\u3c/i\u3e Genes Contribute to Viral Potency

Enhancins are a group of proteins first identified in granuloviruses (GV) that have the ability to enhance nuclear polyhedrosis virus potency. We had previously identified an enhancin gene (E1) in the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) (D. S. Bischoff and J. M. Slavicek, J. Virol. 71:8133–8140, 1997). Inactivation of the E1 gene product within the viral genome lowered viral potency by an average of 2.9-fold. A second enhancin gene (E2) was identified when the entire genome of LdMNPV was sequenced (Kuzio et al., Virology 253:17–34, 1999). The E2 protein exhibits approximately 30% amino acid identity to the LdMNPV E1 protein as well as the enhancins from Trichoplusia ni GV, Pseudaletia unipuncta GV, Helicoverpa armigera GV, and Xestia c-nigrum GV. Northern analysis of viral RNA indicated that the E2 gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The effect of the enhancin proteins on viral potency was investigated through bioassay using two recombinant viruses, one with a deletion in the E2 gene (E2del) and a second with deletion mutations in both enhancin genes (E1delE2del). The enhancin gene viral constructs were verified by Southern analysis and shown not to produce enhancin gene transcripts by Northern analysis. The E2del virus exhibited an average decrease in viral potency of 1.8-fold compared to wild-type virus. In the same bioassays, the recombinant virus E1cat, which does not produce an E1 gene transcript, exhibited an average decrease in viral potency of 2.3-fold compared to control virus. The E1delE2del virus exhibited an average decrease in viral potency of 12-fold compared to wild-type virus. Collectively, these results suggest that both LdMNPV enhancin genes contribute to viral potency, that each enhancin protein can partially compensate for the lack of the other protein, and that both enhancin genes are necessary for wild-type viral potency

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

Época da indução e evocação floral em Citrus sinensis (L.) Osbeck cv. Pêra Rio The flower induction and evocation period in Citrus sinensis (L.) Osbeck cv. Pêra Rio

A produção do estímulo floral (indução) e emissão das primeiras sépalas, na gema floralmente determinada (evocação floral) em Citrus sinensis (L.) Osbeck cv. Pêra Rio pôde ser acompanhada pelas variações anatômicas nos meristemas apicais e axilares. Com objetivo de determinar a época na qual ocorre a indução e evocação floral, cortes longitudinais de gemas apicais e axilares, corados com pironina Y-methylgreen foram efetuados em períodos regulares de maio a agosto. Através das mudanças no formato da gema, principalmente diâmetro, que aumentou com a indução, foi possível determinar a época do ano na qual gemas vegetativas são induzidas a florescerem. Foram detectadas variações nos diâmetros das gemas no período de início da morfogênese floral (meados até o final de julho). A medição do diâmetro das gemas mostra-se um procedimento eficiente para acompanhar os fenômenos decorrentes da indução, evocação e morfogênese florais. Gemas vegetativas apresentam diâmetro médio de 100µm, com túnica composta por três camadas, de formato cônico, recobrindo o corpo, ao passo que a reprodutiva é mais achatada, após a iniciação do primeiro primórdio de sépala, exibindo diâmetro médio de 200µm. Coloração com pironina Y-methylgreen proporciona a captação do fenômeno da evocação floral, pela detecção de regiões mais concentradas em RNA, nas zonas periféricas das gemas; alteração bioquímica esta que precede a emissão das sépalas.<br>The research aims to determine the time in which the inflorescence induction and evocation occurs. The production of inflorescence stimulus (induction) and emission of first sepals in a bud determined as a flower bud (floral evocation) in Citrus sinensis (L.) Osbeck cv. Pêra Rio could be observed through the anatomic variations of the apical and lateral meristems. Longitudinal sections, stained with pironina Y-methylgreen were done in regular periods from May to August. The time of the year in which the vegetative buds are induced to blossom was determined through the bud shape changes, chiefly considering the diameter, which has increased with the induction. Bud diameter variations were detected on the second half of July, the period of floral morphogenesis incipience. The measurement of the bud diameter is an efficient procedure to follow up the phenomena originated from the floral induction, evocation and morphogenesis. Vegetative buds bring out an average diameter of 100µm with a three-layer tunic in a conic shape, recovering its body. The reproductive bud, in turn, is flatter, after the initiation of the first sepal primordium, showing an average diameter of 200µm. The pironina Y-methylgreen stain provides the observation of the floral evocation phenomenon, through the detection of regions with a higher RNA level, in the bud external areas. This biochemical alteration precedes the sepal emission