Morphological properties of the occlusion body of Adoxophyes orana granulovirus.

A granulovirus (GV) that produces occlusion bodies (OBs) having an unusual morphology was found in an Adoxophyes sp. (Lepidoptera: Tortricidae) larva in a tea field in Miyazaki Prefecture, Japan. This isolate is considered to be a mutant of Adoxophyes orana granulovirus, designated AdorGV-M, because the nucleotide sequence of its genome is 99.7% identical to that of an English isolate of AdorGV, AdorGV-E. AdorGV-E produces typical ovocylindrical OBs that contain one occlusion-derived virus (ODV) per OB. On the other hand, AdorGV-M produces large cuboidal OBs, but the number of ODVs per OB was unknown. In this study, we quantified viral DNA in OBs of both AdorGV-E and -M, and determined the number of ODVs occluded in an OB of AdorGV-M. The two isolates had the same quantity of viral DNA in each OB, and we thus confirmed that one OB of AdorGV-M contains one ODV. To investigate the process of OB formation, fat body tissue of A. honmai larvae inoculated with each isolate was observed in a time course by transmission electron microscopy, and OB sizes were measured from micrographs. The main difference in OB formation was that AdorGV-M required more time to mature than AdorGV-E. In AdorGV-E, ODVs began to be covered from one end with an ovocylindrical OB at 96 h post-inoculation (hpi), and most of them were completely occluded at 120 hpi. Occlusion of AdorGV-M ODVs also began at 96 hpi, but the OB shape was cuboidal. Moreover, the OB size of AdorGV-M was similar to that of AdorGV-E at 120 hpi, but continued to grow until 192 hpi. AdorGV-M thus took more time to complete OB formation. Consequently, AdorGV-E has mature OBs with a diameter 0.22 µm and length 0.39 µm, but those of AdorGV-M are 1.34 × 1.23 µm.

Comparison of microscopic and molecular enumeration methods for insect viruses: Cryptophlebia leucotreta granulovirus as a case study.

Enumeration techniques were compared for quantification of the South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA), used as a biopesticide to control false codling moth (Thaumatotibia leucotreta), an insect pest of various fruits and nuts, including citrus. The routine enumeration method for CrleGV-SA virus particles in experimentation and production of CrleGV-SA biopesticides is dark field microscopy. This method was compared with spectrophotometry, scanning electron microscopy (SEM) and real time quantitative polymerase chain reaction (qPCR). The purpose was to develop an accurate and reliable routine enumeration method for CrleGV-SA occlusion bodies (OBs) and to validate the use of dark field microscopy. Purified and semi-purified CrleGV-SA viral stocks were used. Spectrophotometry was not a suitable or accurate enumeration method. Dark field microscopy and SEM were accurate and statistically comparable (p = 0.064), validating the use of dark field microscopy as an enumeration method for granulovirus (GV). However, SEM has superior resolution and the advantage of easily distinguishing virus particles from debris in semi-purified viral stock preparations. A quantitative PCR technique has been developed based on use of specific oligonucleotide primers for the granulin gene. This has the advantage of not being affected by contamination with non-biological debris or biological material, which impact on the other methods.

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

The Complete Genome Sequence of a Second Distinct Betabaculovirus from the True Armyworm, Mythimna unipuncta.

The betabaculovirus originally called Pseudaletia (Mythimna) sp. granulovirus #8 (MyspGV#8) was examined by electron microscopy, host barcoding PCR, and determination of the nucleotide sequence of its genome. Scanning and transmission electron microscopy revealed that the occlusion bodies of MyspGV#8 possessed the characteristic size range and morphology of betabaculovirus granules. Barcoding PCR using cytochrome oxidase I primers with DNA from the MyspGV#8 collection sample confirmed that it had been isolated from the true armyworm, Mythimna unipuncta (Lepidoptera: Noctuidae) and therefore was renamed MyunGV#8. The MyunGV#8 genome was found to be 144,673 bp in size with a nucleotide distribution of 49.9% G+C, which was significantly smaller and more GC-rich than the genome of Pseudaletia unipuncta granulovirus H (PsunGV-H), another M. unipuncta betabaculovirus. A phylogeny based on concatenated baculovirus core gene amino acid sequence alignments placed MyunGV#8 in clade a of genus Betabaculovirus. Kimura-2-parameter nucleotide distances suggested that MyunGV#8 represents a virus species different and distinct from other species of Betabaculovirus. Among the 153 ORFs annotated in the MyunGV#8 genome, four ORFs appeared to have been obtained from or donated to the alphabaculovirus lineage represented by Leucania separata nucleopolyhedrovirus AH1 (LeseNPV-AH1) during co-infection of Mythimna sp. larvae. A set of 33 ORFs was identified that appears only in other clade a betabaculovirus isolates. This clade a-specific set includes an ORF that encodes a polypeptide sequence containing a CIDE_N domain, which is found in caspase-activated DNAse/DNA fragmentation factor (CAD/DFF) proteins. CAD/DFF proteins are involved in digesting DNA during apoptosis.

Optical detection and sizing of single nanoparticles using continuous wetting films.

The physical interaction between nanoscale objects and liquid interfaces can create unique optical properties, enhancing the signatures of the objects with subwavelength features. Here we show that the evaporation on a wetting substrate of a polymer solution containing submicrometer or nanoscale particles creates liquid microlenses that arise from the local deformations of the continuous wetting film. These microlenses have properties similar to axicon lenses that are known to create beams with a long depth of focus. This enhanced depth of focus allows detection of single nanoparticles using a low-magnification microscope objective lens, achieving a relatively wide field-of-view, while also lifting the constraints on precise focusing onto the object plane. Hence, by creating these liquid axicon lenses through spatial deformations of a continuous thin wetting film, we transfer the challenge of imaging individual nanoparticles to detecting the light focused by these lenses. As a proof of concept, we demonstrate the detection and sizing of single nanoparticles (100 and 200 nm), CpGV granuloviruses, as well as Staphylococcus epidermidis bacteria over a wide field-of-view of 5.10 × 3.75 mm(2) using a 5× objective lens with a numerical aperture of 0.15. In addition to conventional lens-based microscopy, this continuous wetting-film-based approach is also applicable to lens-free computational on-chip imaging, which can be used to detect single nanoparticles over a large field-of-view of >20-30 mm(2). These results could be especially useful for high-throughput field analysis of nanoscale objects using compact and cost-effective microscope designs.

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.

ODV-associated proteins of the Pieris rapae granulovirus.

Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus--host interaction studies.

Characterization and partial genome sequence analysis of Clostera anachoreta granulovirus.

The morphological and biological properties as well as partial genomic sequencing of a granulovirus isolated from Clostera anachoreta (Lepidoptera: Notodontidae), C. anachoreta granulovirus (ClanGV), were carried out. The ovoidal occlusion bodies were 337 nm x 170 nm in size, and each granule contained one single rod-shape virion, with a mean size of 250 nm x 46 nm. Granulin had a molecular weight of approximately 30 kDa. ClanGV genome size was estimated as 104.34 kb based on the restriction fragments. The restriction pattern of the ClanGV genome was different from other GVs. A restriction fragment genomic library of ClanGV genome was constructed. The library consisted of nine SalI fragments, seven HindIII fragments and seven EcoRI fragments. One 4.8 kb fragment of the genome, digested by SalI, was sequenced and analyzed. This region was composed of eight unknown ORFs, two baculoviruses homologous gene (vp1054 and lef10) and partial sequence of lef-8. The unknown ORFs included three unique to ClanGV, the other five ORFs were related to baculoviruses. The ORFs, located within this restriction fragment, were compared to homologues in other GVs. The results indicated that ClanGV, CpGV, ClGV, AoGV and PoGV had similar arrangement and orientation of the homologous ORFs. Phylogenetic analysis of VP1054 proteins from 20 baculoviruses indicated that ClanGV was more closely related to CpGV, ClGV, AoGV and PoGV than to other baculoviruses.

Discovery of a granulovirus of Pandemis pyrusana (Lepidoptera: Tortricidae), a leafroller pest of apples in Washington.

Pandemis pyrusana (Kearfott) is an important leafroller pest of apples in Washington. Surveys for natural enemies discovered a pathogen infecting Pandemis leafrollers in an apple orchard in central Washington. The pathogen was propagated in the laboratory and light microscopy using an azan stain demonstrated that it infected fat body, epidermis, and tracheal matrix cells. The virus was identified morphologically as a granulovirus using electron microscopy and designated PpGV. Rates of infection were determined for each generation in an apple orchard for three years. Infection rates were variable and ranged from 2.6 to 67% of individuals collected from each generation.

Effects of the Epap granulovirus on its host, Epinotia aporema (Lepidoptera: Tortricidae).

The bean shoot borer, Epinotia aporema, is a major pest of soybeans in Argentina. Larvae of this pest are attacked by a granulovirus (EpapGV) that is the most important cause of sporadic epizootics in E. aporema populations. We studied the pathology of this virus in last-instar larvae using light and electron microscopy, and evaluated the effect of the disease on larval growth and development. EpapGV caused a polyorganotropic infection. No nucleocapsids were observed in the nuclei of infected cells prior to nuclear membrane disruption. Nevertheless, granulin was detected in the nucleus by immuno-gold staining, indicating that late gene expression occurred prior to nuclear membrane disruption. Establishment of the virogenic stroma led to complexes of continuous parallel convoluted membranous sheets. Nucleocapsids were enveloped in these areas to form virions, which were then occluded. Apparently as part of the cell-to-cell spread of infection, nucleocapsids were observed enclosed in large numbers within membrane-bound vesicles located between the cells and basal lamina. Larvae infected by EpapGV suffered a retardation of development and typically failed to pupate, but exhibited a weight increase greater than that of healthy E. aporema.