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Granuloviruses are insect viruses commonly used as biological insecticides for pest control in agriculture. Most pest control programs rely on the most virulent strains and doses to cause acute mortality events. However, we hypothesize that sublethal infections are key to maintaining persistent epidemics that regulate pest populations in the long‐term. The tomato leafminer (TLM), Tuta absoluta (Meyrick) (= Phthorimaea absoluta Meyrick) (Lepidoptera: Gelechiidae), is one of the most limiting pests of greenhouse and field tomatoes. Previous efforts have identified a strain of Phthorimaea operculella granulovirus (PhopGV), coded VG013, associated with field populations of the TLM, which causes significant acute mortalities and is being used as active ingredient of a biological insecticide. The aim of this study was to determine the effect of sublethal infections and transmission mechanisms of PhopGV‐VG013 on TLM populations. Six viral doses were tested for their virulence and effects on TLM development rate, and intrinsic population growth rate. A sublethal dose was selected to compare the density‐dependent mortality of TLM populations and vertical transmission was examined by testing for PhopGV in the offspring of surviving infected individuals. Horizontal transmission was assessed by testing for PhopGV in frass of infected larvae, and the infection of virus‐free larvae confined with infected larvae in the same leaf. We found that sublethal doses of PhopGV have slight effects on TLM population growth rates, and none on the strength of density‐dependent mortality. Although we found for the first time that PhopGV‐VG013 is transmitted to approximately 7.5% of the offspring of individuals that survive the virus challenge, we found no evidence suggesting horizontal transmission from sublethal infections. Our results show that sublethal infections of highly virulent ganulovirus strains may favor long‐term pathogen persistence but have a limited role in host population regulation. [ABSTRACT FROM AUTHOR]

Baculoviruses are DNA viruses that can naturally control insects and form the basis for a range of biological insecticides. These viruses are occluded in a crystalline protein matrix, the occlusion body (OB), which favors their persistence in the environment until consumed by a susceptible insect. This review presents evidence that baculoviruses are perfectly adapted to persist in soil. In support of this assertion, detailed evidence is presented concerning the stability of virus OBs in soil, the mechanisms and processes that affect OB transport to soil, OB persistence and movement through the soil layers, and the transport of the virus back to the host's foodplant for subsequent transmission. The soil reservoir interacts with the host insect, the foodplant and the biotic and abiotic factors in the environment, all of which influence the function of the soil OB reservoir. Agricultural practices that conserve the soil reservoir can result in improved pest control. The soil is also an overlooked source of genetic diversity for the design of baculovirus-based insecticides. Finally, the principal issues that remain to be elucidated are highlighted, particularly with reference to those that affect the soil–baculovirus relationship and the impact of the soil OB reservoir on insect populations. [ABSTRACT FROM AUTHOR]

The cabbage butterfly, Pieris brassicae (L.) is an economically important cosmopolitan insect, that causes appreciable crop loss in cabbage, cauliflower and other cruciferous crops, especially during winter and early spring in India. To reduce its pest density, a granulovirus infecting Pieris brassicae (PibrGV) was found to be a promising eco-friendly pathogen in the present study. Our report describes the pathogenicity, structural details, genome sequence, and characterization of the PibrGV Manipur isolate. The median lethal concentration and survival time were calculated for the third instar larvae of P. brassicae and the bioassay revealed that LC50 and LT50 of the virus were found to be 4.8 x 104 OBs/ml and 120 hours, respectively. Post-purification of occlusion bodies, the viral DNA was extracted and subjected to genome sequencing and annotation. There were 128 Glimmer-predicted ORFs in the 106,469 bp long viral genome characterized by 33.7% GC content. The study further indicated that a total of 14 genes were involved in replication, 10 genes were associated with transcription and 24 genes were related to structure formation, respectively. The above database will help in understanding the molecular mechanisms and genetic makeup of the virus and assist in pest management strategies through recombinant DNA technology. [ABSTRACT FROM AUTHOR]

The potential for immigrant adult codling moths, Cydia pomonella (L.), to reduce the efficacy of the granulovirus (CpGV) against a univoltine population was investigated using published life tables for codling moth on apple trees in Nelson, New Zealand. Simulated life tables over eight generations incorporated mortality >90% from CpGV sprays and combined this with immigration rates ranging from 0 to 1 adult per tree. Without immigration, there were 5 damaged fruits per million by the eighth generation from an initial population of 1.22 adults per tree but only 9 per 100 million had fresh damage or larvae that could trigger phytosanitary rejection. Higher-yielding modern orchards reduced this fresh damage to 5 per 100 million harvested fruit. New Zealand exports apples to markets with zero tolerance of codling moth (Codling Moth Sensitive Markets [CMSM] programme), and immigration of 1 female per 100 trees was projected to pose a risk of failure to meet quarantine requirements with one freshly damaged fruit per 112,000, or per 202,000 in a modern high-density orchard. This confirms the wisdom of using an integrated suite of control methods in CMSM, of which CpGV is only one. Similarly, immigration of one female per 1000 trees or 3 ha had much lower risk but could also threaten exporting if reliance was placed solely on CpGV, with one fresh damage or larva per 2 million harvested fruit. A commitment to the removal of neglected host trees of codling moth in the environs of orchards is recommended. [ABSTRACT FROM AUTHOR]

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Baculoviruses are DNA viruses that can naturally control insects and form the basis for a range of biological insecticides. These viruses are occluded in a crystalline protein matrix, the occlusion body (OB), which favors their persistence in the environment until consumed by a susceptible insect. This review presents evidence that baculoviruses are perfectly adapted to persist in soil. In support of this assertion, detailed evidence is presented concerning the stability of virus OBs in soil, the mechanisms and processes that affect OB transport to soil, OB persistence and movement through the soil layers, and the transport of the virus back to the host’s foodplant for subsequent transmission. The soil reservoir interacts with the host insect, the foodplant and the biotic and abiotic factors in the environment, all of which influence the function of the soil OB reservoir. Agricultural practices that conserve the soil reservoir can result in improved pest control. The soil is also an overlooked source of genetic diversity for the design of baculovirus-based insecticides. Finally, the principal issues that remain to be elucidated are highlighted, particularly with reference to those that affect the soil–baculovirus relationship and the impact of the soil OB reservoir on insect populations.

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.

Plutella xylostella (Lepidoptera: Plutellidae) is a cosmopolitan pest, infesting the cruciferous crops worldwide. It has developed resistance against chemical insecticides. To overcome this problem there is dire need to explore alternative control measures which are safer to the environment and compatible with human health. The present study was conducted to investigate the insecticidal properties of native isolated Plutella xylostella granulovirus (PxGV) and Azadirachta indica (AZA) on mortality and development of P. xylostella under laboratory conditions. Both AZA and PxGV were applied alone and in integrated manners at LC20 and LC50 dose rates. The combination of 349.3 parts per million of AZA and 4.8x104 occlusion bodies per millimeter of PxGV, resulted in synergistic interaction after 3rd, 5th and 8th days post application while the additive effect was observed only after 1 day post-treatment. Moreover, combined application of PxGV and AZA at sub-lethal doses exhibited increased larval, pupal and adult longevity and decreased larval and adult weight. In conclusion, combination of PxGV and AZA at low dose rate is a viable option to control diamondback moth. [ABSTRACT FROM AUTHOR]

The fall webworm (Hyphantria cunea) impacts a wide variety of crops and cultivated broadleaf plant species. The pest is native to North America, was introduced to Europe and has since spread further as far as central Asia. Despite several attempts to control its distribution, the pest continues to spread causing damage all over the world. A naturally occurring baculovirus, Hyphantria cunea granulovirus (HycuGV-Hc1), isolated from the larvae of H. cunea in Turkey appears to have a potential as microbial control agent against this pest. In this report we describe the complete genome sequence and organization of the granulovirus isolate (HycuGV-Hc1) that infects the larval stages and compare it to other baculovirus genomes. The HycuGV-Hc1 genome is a circular double-stranded DNA of 114,825 bp in size with a nucleotide distribution of 39.3% G + C. Bioinformatics analysis predicted 132 putative open reading frames of (ORFs) ≥ 150 nucleotides. There are 24 ORFs with unknown function. Seven homologous repeated regions (hr s) and two bro genes (bro -1 and bro -2) were identified in the genome. Comparison to other baculovirus genomes, HycuGV-Hc1 revealed some differences in gene content and organization. Gene parity plots and phylogenetics confirmed that HycuGV-Hc1 is a Betabaculovirus and is closely related to Plutella xylostella granulovirus. This study expands our knowledge on the genetic variation of HycuGV isolates and provides further novel knowledge on the nature of granuloviruses. • The HycuGV-Hc1 DNA genome has 114,825 bp, GC content of 39.3% and 132 putative ORFs. • Seven homologous regions (hrs) were identified in the genome. • The phylogenetic tree showed that HycuGV-Hc1 to be closely related to the PlxyGV. • HycuGV-Hc1 is infective for H. cunea larvae which impacts a wide variety of crops. [ABSTRACT FROM AUTHOR]

Metagenomic analysis of Aedes aegypti and Ae. albopictus mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant reads found in Ae. aegypti mosquitoes were of Phasi Charoen-like virus (PCLV), Choristoneura fumiferana granulovirus (CfGV), Cell fusing agent virus (CFAV), and Wenzhou sobemo-like virus 4 (WSLV4), whereas WSLV4 and CfGV constituted the highest percentage of reads in Ae. albopictus viromes. Other reads that were of low percentage included Hubei mosquito virus 2 (HMV2), Porcine astrovirus 4 (PAstV4), and Wild Boar astrovirus (WBAstV). PCLV and CFAV, which were found to be abundant in Ae . aegypti viromes were absent in Ae. albopictus viromes. Among the viromes analyzed, Ae. aegypti sampled from Pune showed the highest percentage (79.82%) of viral reads, while Ae . aegypti mosquitoes sampled from Dibrugarh showed the lowest percentage (3.47%). Shamonda orthobunyavirus (SHAV), African swine fever virus (ASFV), Aroa virus (AROAV), and Ilheus virus (ILHV), having the potential to infect vertebrates, including humans, were also detected in both mosquito species, albeit with low read numbers. Reads of gemykibivirus, avian retrovirus, bacteriophages, herpesviruses, and viruses infecting protozoans, algae, etc., were also detected in the mosquitoes. A high percentage of reads in the Ae. albopictus mosquito samples belonged to unclassified viruses and warrant further investigation. The data generated in the present work may not only lead to studies to explain the influence of these viruses on the replication and transmission of viruses of clinical importance but also to find applications as biocontrol agents against pathogenic viruses.

Background: The hornworn Erinnyis ello is the major pest of natural rubber crops in Colombia, mainly controlled using toxic chemical insecticides. The use of E. ello Betabaculovirus is an environmentally sustainable alternative for its control. The aim of the present work was to characterize a prototype biopesticide formulation and evaluate its efficacy under different conditions. Results: Quality control evaluations of formulated biopesticide revealed that all the parameters evaluated were under the permissible level. The lethal concentrations LC50 and LC90 of the biopesticide were 4.3 × 103 and 5.5 × 104 occlusion bodies (OBs) mL−1, respectively. Biopesticide efficacies against second and fourth instar larvae under greenhouse conditions were higher than 80%. Evaluation of two application rates in a clonal garden resulted in 84% and 88% efficacy, comparable to that obtained with the chemical. The biopesticide in a commercial plantation showed efficacies between 74% and 82%. Biopesticide post‐application persistence was estimated at least in 1 week under field natural conditions. Results allowed selection of the lowest evaluated dose (1 × 1011 OBs ha−1) as the basis for further field evaluations. Conclusion: Formulated ErelGV showed high efficacy to control the hornworm in rubber crops and high potential to be included in integrated pest management programs, thus it could be an interesting alternative to replace agrochemicals. © 2018 Society of Chemical Industry Erinnyis ello is the major pest of rubber crops in Colombia, mainly controlled using toxic agrochemicals. This article presents the use of a betabaculovirus as a sustainable control alternative. [ABSTRACT FROM AUTHOR]

Matsumuraeses phaseoli is a Lepidopteran pest that primarily feeds on numerous species of cultivated legumes, such as Glycine and Phaseolus. It is widely distributed in northeast Asia. A novel granulovirus, designated as Matsumuraeses phaseoli granulovirus (MaphGV), was isolated from pathogenic M. phaseoli larvae that dwell in rolled leaves of Astragalus membranaceus, a Chinese medicinal herb. In this study, using next-generation sequencing, we report the complete genome of MaphGV. MaphGV genome comprises a double-stranded DNA of 116,875 bp, with 37.18% GC content. It has 128 hypothetical open reading frames (ORFs). Among them, 38 are baculovirus core genes, 18 are lepidopteran baculovirus conserved genes, and 5 are unique to Baculoviridae. MaphGV has one baculovirus repeat ORF (bro) and three inhibitors of apoptosis proteins (iap), including a newfound iap-6. We found two atypical baculoviral homologous regions (hrs) and four direct repeats (drs) in the MaphGV genome. Based on phylogenetic analysis, MaphGV belongs to Clade b of Betabaculovirus and is closely related to Cydia pomonellagranulovirus (CpGV) and Cryptophlebia leucotretagranulovirus (CrleGV). This novel baculovirus discovery and sequencing are invaluable in understanding the evolution of baculovirus and MaphGV may be a potential biocontrol agent against the bean ravaging pest.

Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.

A broad survey of the fall webworm (Lepidoptera: Erebidae) populations in agricultural and forested areas in the Central Black Sea Region of Turkey led to the detection of a granulovirus (GV). Hyphantria cunea granulovirus (HycuGV)-Hc1 isolate was characterized and tested against third instar larvae of H. cunea. Electron microscopy confirmed typical GV morphology with ovoid granules of approximately 368 ±  16 nm × 201 ±  19 nm. Each granule contained a single rod-shaped virion with a mean size of 43 ± 12 nm × 250 ± 12 nm. The genome was analyzed by restriction endonuclease and estimated to be ∼112 kb. Partial sequencing of the granulin (gran), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes also confirmed the identity of the virus as HycuGV. A phylogenetic analysis based on these conserved genes, HycuGV-Hc1 grouped together with the previous HycuGV isolate (A5-1) and Estigmene acrea granulovirus (EsacGV) isolate from the same family. The LC50 of HycuGV-Hc1 was 2.6 × 104 occlusion bodies (OBs/ml). Pot experiments, under field conditions, showed significant differences between virus treated and control groups. This is the first study to describe a novel Turkish HycuGV-Hc1 isolate, and preliminary data suggests that the virus has a significant potential as an effective biopesticide for control of H. cunea. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Heavy infestations of stored potato (Solanum tuberosum L.) tubers by the two potato tuber moth species Symmetrischema tangolias (Gyen) and Tecia solanivora (Povolny) frequently occur in Andean potato‐growing regions of Ecuador. The aim of the study was to develop a biological control strategy for both species using powder formulations made of inert substances, Phthorimaea operculella (Zeller) granulovirus (PhopGV) and Bacillus thuringiensis Berliner subsp. kurstaki (Btk). The LC50 of PhopGV on T. solanivora was 0.33 LE/L, and Btk caused 82.7% mortality at a concentration of 100 g/L in bioassays. The efficacy of talcum, kaolin, calcium carbonate and sand ranged between 76.2% and 98.7%. Calcium carbonate was highly effective to control both species; however, its efficacy was affected by the relative humidity and dropped to 55.4% at relative humidity of 100%. PhopGV at concentrations of five larvae equivalents (LE) per kg kaolin and Btk at a concentration of 60 g Btk/kg talcum caused 95.7% and 88.1% mortality of T. solanivora, respectively. In storage experiments, the efficacy of calcium carbonate alone and in combination with PhopGV (20 LE/kg) and Btk (15 g/kg) caused 95.0–99.8% mortality of T. solanivora in all treatments and reduced infestation on potato tubers by 83.6%–91.0%. In the case of S. tangolias, Btk significantly increased mortality to 96.5% compared with calcium carbonate alone and reduced tuber infestation by 83.4%. Storage of potato tubers in thin layers enhanced the efficacy of the calcium carbonate treatment compared with storage in bags. It was concluded that calcium carbonate alone seems to be appropriate for the control of T. solanivora, and an addition of 15 g Btk/kg would improve the control of S. tangolias. It is suggested to test these new formulations under on‐farm storage conditions. [ABSTRACT FROM AUTHOR]

The ability of the isolate VG008 of S. frugiperda granulovirus (SpfrGV) to enhance the infectivity of the isolate SfCOL of S. frugiperda multiple nucleopolyhedrovirus (SpfrMNPV) was evaluated on S. frugiperda larvae. Bioassays were performed with mixtures by using different proportions 90%:10% (M1), 95%:5% (M2) and 97.5%:2.5% (M3) of SfCOL:VG008, respectively. All mixtures showed higher insecticidal activity that SfCOL. The mixture M3 showed the highest enhancement of SfCOL reducing 11.40 times the Mean Lethal Concentration and 96 h in the Mean Time to Death. The enhancer activity of proteins derived from VG008 (GVPs) were also evaluated in mixture with SfCOL. The GVPs increased 27% larval mortality caused by SfCOL and damaged the peritrophic membrane of S. litura larvae, suggesting that the key point in this enhancing activity is the initial step of the larva colonization, the midgut infection. M3 was formulated and evaluated under greenhouse conditions in maize plants using different doses. The highest efficacy was obtained with the highest dose of M3 (8 × 1011 OBs/ha), which was similar to that found when formulated SfCOL was applied using an approximately twofold higher dose. The viral mixture M3 was selected as the active ingredient for developing a new biopesticide for a more efficient management of the pest in the field.

Achaea janata granulovirus (AcjaGV), an insect virus belonging to Baculoviridae, infects semilooper, a widely distributed defoliating pest on castor beans ( Ricinus communis L.) and several other plant hosts in India. The propagation and purification of the Hyderabad isolate AcjaGV were performed, granulin gene from this isolate was amplified, cloned and sequenced, and its homology with other known granulin genes was assessed. The 753-bp granulin ORF of AcjaGV encoded for a granulin protein of 250 amino acids with a molecular mass of 29.5 ± 0.7 kDa. This amino acid sequence exhibited significant homology with Spodoptera litura granulovirus (SpliGV) and other GVs infecting insects in the same Noctuidae family of Lepidoptera. Peptide analysis of granulin protein indicated close homology with that of SpliGV. Virtual RFLP patterns from in silico digestions of granulin gene of 18 granuloviruses mapped by 12 restriction enzymes were used for simulated digestions. Implications of the phylogenetic relationships of granulin nucleotide and deduced amino acid sequence are discussed. We have established the sequence identity of granulin gene of AcjaGV and characterized its protein product and the phylogenetic relationship with other known GVs. Our results indicate the presence of unique restriction sites for three restriction enzymes, and this can be used as a tool for identification of AcjaGV from various sources. This is the first report from the Indian subcontinent to describe the complete granulin gene of a GV isolated from A. janata. [ABSTRACT FROM AUTHOR]

The use of Cydia pomonella granulovirus (CpGV) isolates as biological control agents of codling moth (CM) larvae is important in organic and integrated pome fruit production worldwide. The commercially available isolates CpGV-0006, CpGV-R5, and CpGV-V15 have been selected for the control of CpGV resistant CM populations in Europe. In infection experiments, CpGV-0006 and CpGV-R5 were able to break type I resistance and to a lower extent also type III resistance, whereas CpGV-V15 overcame type I and the rarely occurring type II and type III resistance. The genetic background of the three isolates was investigated with next generation sequencing (NGS) tools by comparing their nucleotide compositions to whole genome alignments of five CpGV isolates representing the known genetic diversity of the CpGV genome groups A to E. Based on the distribution of single nucleotide polymorphisms (SNPs) in Illumina sequencing reads, we found that the two isolates CpGV-0006 and CpGV-R5 have highly similar genome group compositions, consisting of about two thirds of the CpGV genome group E and one third of genome group A. In contrast, CpGV-V15 is composed of equal parts of CpGV genome group B and E. According to the identified genetic composition of these isolates, their efficacy towards different resistance types can be explained and predictions on the success of resistance management strategies in resistant CM populations can be made. [ABSTRACT FROM AUTHOR]

About 20,550 larvae, pupae and adults of the codling moth, Cydia pomonella L., were diagnosed for pathogens during long-term investigations (1955–2012) at the Institute for Biological Control in Darmstadt, Germany. The prevailing entomopathogens diagnosed in these studies were insect pathogenic fungi, especially Beauveria bassiana and Isaria farinosa, the microsporidium, Nosema carpocapsae, the Cydia pomonella granulovirus (CpGV), as well as mostly undetermined bacteria. While the CpGV was observed exclusively in larvae and pupae from laboratory colonies or from field experiments with this virus, entomopathogenic fungi were most frequently diagnosed in last instars in autumn and in diapausing larvae and pupae in spring. B. bassiana was identified as the major fungal pathogen, causing larval prevalences of 0.9% to 100% (mean, about 32%). During prognostic long-term studies in larvae and adults of C. pomonella, N. carpocapsae was diagnosed in codling moth populations from various locations in Germany. The mean prevalence generally ranged between 20% and 50%. Experiments revealed that the fecundity and fertility of microsporidia-infected female adults were significantly reduced compared to healthy ones. The results underpin the importance of naturally occurring microbial antagonists and represent a base for further ecological studies on developing new or additional biological and integrated control strategies.

The Baculoviridae, a family of insect-specific large DNA viruses, is widely used in both biotechnology and biological control. Its applied value stems from millions of years of evolution influenced by interactions with their hosts and the environment. To understand how ecological interactions have shaped baculovirus diversification, we reconstructed a robust molecular phylogeny using 217 complete genomes and ~580 isolates for which at least one of four lepidopteran core genes was available. We then used a phylogenetic-concept-based approach (mPTP) to delimit 165 baculovirus species, including 38 species derived from new genetic data. Phylogenetic optimization of ecological characters revealed a general pattern of host conservatism punctuated by occasional shifts between closely related hosts and major shifts between lepidopteran superfamilies. Moreover, we found significant phylogenetic conservatism between baculoviruses and the type of plant growth (woody or herbaceous) associated with their insect hosts. In addition, we found that colonization of new ecological niches sometimes led to viral radiation. These macroevolutionary patterns show that besides selection during the infection process, baculovirus diversification was influenced by tritrophic interactions, explained by their persistence on plants and interactions in the midgut during horizontal transmission. This complete eco-evolutionary framework highlights the potential innovations that could still be harnessed from the diversity of baculoviruses.

Viruses of four families of arthropod-specific, large dsDNA viruses (the nuclear arthropod large DNA viruses, or NALDVs) possess homologs of genes encoding conserved components involved in the baculovirus primary infection mechanism. The presence of such homologs encoding per os infectivity factors (pif genes), along with their absence from other viruses and the occurrence of other shared characteristics, suggests a common origin for the viruses of these families. Therefore, the class Naldaviricetes was recently established, accommodating these four families. In addition, within this class, the ICTV approved the creation of the order Lefavirales for three of these families, whose members carry homologs of the baculovirus genes that code for components of the viral RNA polymerase, which is responsible for late gene expression. We further established a system for the binomial naming of all virus species in the order Lefavirales, in accordance with a decision by the ICTV in 2019 to move towards a standardized nomenclature for all virus species. The binomial species names for members of the order Lefavirales consist of the name of the genus to which the species belongs (e.g., Alphabaculovirus), followed by a single epithet that refers to the host species from which the virus was originally isolated. The common names of viruses and the abbreviations thereof will not change, as the format of virus names lies outside the remit of the ICTV., (© 2023. The Author(s).)

A baculovirus, Cnaphalocrocis medinalis (Guenée) granulovirus (CnmeGV) was isolated from the infected caterpillars of the rice leaf folder, Cnaphalocrocis medinalis Güenée (Lepidoptera: Pyralidae) and was analyzed in biological and molecular characterizations. The occlusion body of CnmeGV was in ovoidal shape, with an average size of 384 x 211nm. Each occlusion body contained one single rod-shape virion with a mean size of 237 nm in length taband 39 nm in width. Occlusion body matrix analysis indicated that, despite the molecular weight of 31 kD granulins, there was another special protein lane for molecular mass about 14.5 kD. Phylogenetic analysis based on amino acid sequences of gran, lef-8, and lef-9 revealed that CnmeGV is grouped in the Betabaculovirus genus. Restriction endonuclease fragment analysis showed that the total genome size of CnmeGV was about 113 kb. Bioassay test at the concentration of 2.5×103 OBs/g of the CnmeGV showed the killing efficacy to different instar larvae ranged from 30.7 to 51.4% in 12 days and 54.8 to 71.3% in 18 days. The median lethal time ranged from 13.2 days in 1st instar larvae to 16.1 days in 3rd instar larvae at the same concentration. The results revealed a promising use of CnmeGV in the future as a potential microbial agent against the rice leaf folder. [ABSTRACT FROM AUTHOR]

Infection of parasitized Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae by a granulovirus (GV) deleteriously affected the development of the endoparasitoid Chelonus inanitus (Linnaeus) (Hymenoptera: Braconidae). When second-instar parasitized S. litura larvae were inoculated with GV, the percentages of emergence, pupation, and adult eclosion of C. inanitus were significantly lower from GV-infected hosts than those from uninfected ones. Lower parasitoid emergence was due to the earlier death of parasitized and GV-infected hosts and slower development of parasitoid larvae in GV-infected hosts than in uninfected ones. Some of the parasitoid larvae that emerged from GV-infected hosts died before pupation and adult eclosion as a result of lower external feeding on GV-infected cadavers. Furthermore, the body volume of parasitoids developing in GV-infected hosts was significantly lower than that of parasitoids developing in uninfected hosts. The experiment in which host cadavers and emerged parasitoids were switched revealed that external feeding of parasitoids was affected both by infection of host cadavers with GV and also by parasitoid development within GV-infected hosts. Our data show that GV infection of S. litura larvae has deleterious effects on C. inanitus development. [ABSTRACT FROM AUTHOR]

The use of Cydia pomonella granulovirus (CpGV) isolates as biological control agents of codling moth (CM) larvae is important in organic and integrated pome fruit production worldwide. The commercially available isolates CpGV-0006, CpGV-R5, and CpGV-V15 have been selected for the control of CpGV resistant CM populations in Europe. In infection experiments, CpGV-0006 and CpGV-R5 were able to break type I resistance and to a lower extent also type III resistance, whereas CpGV-V15 overcame type I and the rarely occurring type II and type III resistance. The genetic background of the three isolates was investigated with next generation sequencing (NGS) tools by comparing their nucleotide compositions to whole genome alignments of five CpGV isolates representing the known genetic diversity of the CpGV genome groups A to E. Based on the distribution of single nucleotide polymorphisms (SNPs) in Illumina sequencing reads, we found that the two isolates CpGV-0006 and CpGV-R5 have highly similar genome group compositions, consisting of about two thirds of the CpGV genome group E and one third of genome group A. In contrast, CpGV-V15 is composed of equal parts of CpGV genome group B and E. According to the identified genetic composition of these isolates, their efficacy towards different resistance types can be explained and predictions on the success of resistance management strategies in resistant CM populations can be made.

Due to its outstanding suitability to produce complex biopharmaceutical products including virus-like particles and subunit vaccines, the baculovirus/insect cell expression system has developed into a highly popular production platform in the biotechnological industry. For high productivity, virus-cell communication and an efficient spreading of the viral infection are crucial, and, in this context, extracellular vesicles (EVs) might play a significant role. EVs are small particles, utilized by cells to transfer biologically active compounds such as proteins, lipids as well as nucleic acids to recipient cells for intracellular communication. Studies in mammalian cells showed that the release of EVs is altered in response to infection with many viruses, ultimately either limiting or fostering infection spreading. In this study we isolated and characterized EVs, from both uninfected and baculovirus infected Tnms42 insect cells. Via quantitative proteomic analysis we identified more than 3000 T. ni proteins in Tnms42 cell derived EVs, of which more than 400 were significantly differentially abundant upon baculovirus infection. Subsequent gene set enrichment analysis revealed a depletion of proteins related to the extracellular matrix in EVs from infected cultures. Our findings show a significant change of EV protein cargo upon baculovirus infection, suggesting a major role of EVs as stress markers. Our study might serve in designing new tools for process monitoring and control to further improve biopharmaceutical production within the baculovirus/insect cell expression system., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hausjell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)