Genomic analyses of a new baculovirus isolated from the wheat armyworm, Mythimna sequax (Franclemont) (Lepidoptera: Noctuidae).

We report the genomic analysis of a novel alphabaculovirus, Mythimna sequax nucleopolyhedrovirus isolate CNPSo-98 (MyseNPV-CNPSo-98), obtained from cadavers of the winter crop pest, Mythimna sequax Franclemont (Lepidoptera: Noctuidae). The insects were collected from rice fields in Southern Brazil in the 1980's and belongs to the 'EMBRAPA-Soja' Virus Collection. High-throughput sequencing reads of DNA from MyseNPV occlusion bodies and assembly of the data yielded an AT-rich circular genome contig of 148,403 bp in length with 163 annotated opening reading frames (ORFs) and four homologous regions (hrs). Phylogenetic inference based on baculovirus core protein sequence alignments indicated that MyseNPV-CNPSo-98 is a member of Alphabaculovirus genus that clustered with other group II noctuid-infecting baculoviruses, including viruses isolated from Helicoverpa armigera and Mamestra spp. The genomes of the clade share strict collinearity and high pairwise nucleotide identity, with a common set of 149 genes, evolving under negative selection, except a bro gene. Branch lengths and Kimura-2-parameter pairwise nucleotide distances indicated that MyseNPV-CNPSo-98 represents a distinct lineage that may not be classified in any of the currently listed species in the genus.

Three picorna-like viruses found associated with the spider mite, Tetranychus truncatus (Acari: Tetranychidae).

Herbivorous arthropods, such as mites and insects, host a variety of microorganisms that significantly influence their ecology and evolution. While insect viruses have been extensively studied, our understanding of the diversity and composition of mite viromes and the interactions with mite hosts remains limited. The Asian spider mite, Tetranychus truncatus Ehara (Acari: Tetranychidae), a major agricultural pest, has not yet been reported to harbor any viruses. Here, using publicly available RNA-Seq data, we identified and characterized three picorna-like viruses associated with T. truncatus: Tetranychus truncatus-associated iflavirus 1 (TtAIV-1), Tetranychus truncatus-associated picorna-like virus 1 (TtAV-1), and Tetranychus truncatus-associated picorna-like virus 2 (TtAV-2). TtAIV-1 has a typical Iflaviridae genome structure with a single ORF, representing the first iflavirus associated with the Tetranychus genus. TtAV-1 and TtAV-2 exhibit bicistronic arrangements similar to dicistroviruses and other picorna-like viruses, with complex secondary structures in their non-coding regions. Phylogenetic analysis places TtAIV-1 within Iflaviridae, possibly as a new species, while TtAV-1 and TtAV-2 form distinct clades within unclassified picorna-like viruses, suggesting new families within Picornavirales. We analyzed in silico the presence and abundance of these viruses in T. truncatus across four bioproject SRAs, mostly finding them co-associated, with viral reads reaching up to 30% of total reads. Their presence and abundance varied by mite treatment and origin, with no significant impact from Wolbachia infection or abamectin exposure, although TtAV-2 was absent in abamectin-treated mites. Temperature influenced virus abundance, and variations were observed among Chinese mite populations based on geography and host plant association. Our findings offer insights into picorna-like virus diversity and dynamics in T. truncatus, revealing potential roles in mite biology and suggesting applications for mite population control, thereby enhancing agricultural productivity and food security.

Easily purified baculovirus/insect-system-expressed recombinant hepatitis B virus surface antigen fused to the N- or C-terminus of polyhedrin.

Baculoviruses are circular double-stranded DNA viruses that infect insects and are widely used as the baculoviral expression vectors (BEVs), which provide a eukaryotic milieu for heterologous expression. The most frequently used vector is based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, purification of recombinant proteins produced using BEVs is laborious, time-consuming, and often expensive. Numerous strategies have been explored to facilitate purification of heterologous proteins, such as fusion with occlusion body (OBs)-forming proteins like polyhedrin (Polh). Baculoviruses produce OBs in the late stages of infection to protect the virion in the cellular environment, and the main protein responsible for OB formation is Polh. In this study, we investigated the effect of fusing the gene that encodes the surface antigen (S-HBsAg) of hepatitis B virus (HBV) to either the N- or C-terminus of the AcMNPV Polh. The production of recombinant viruses and recombinant proteins was confirmed, and the ability to form chimeric S-HBsAg-containing OBs was accessed by light and scanning electron microscopy of infected cells. The fusion was found to affect the shape and size of the OBs when compared to wild-type OBs, with the N-terminal fusion producing less-amorphous OBs than the C-terminal construct. In addition, the N-terminal construct gave higher levels of expression than the C-terminal construct. Quantitative and qualitative immunoassays with human serum or plasma antibodies against HBsAg showed that the two forms of the antigen reacted differently. Although both reacted with the antibody, the N-terminal fusion protein reacted with more sensitivity (2.27-fold) and is therefore more suitable for quantitative assays than the C-terminal version. In summary, the BEVs represents a promising tool for the production of reagents for the diagnosis of HBV infection.

Characterization of a novel alphabaculovirus isolated from the Southern armyworm, Spodoptera eridania (Cramer, 1782) (Lepidoptera: Noctuidae) and the evolution of odv-e66, a bacterium-acquired baculoviral chondroitinase gene.

The Southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae) is native to the American tropics and a polyphagous pest of several crops. Here we characterized a novel alphabaculovirus isolated from S. eridania, isolate Spodoptera eridania nucleopolyhedrivurus CNPSo-165 (SperNPV-CNPSo-165). SperNPV-CNPSo-165 occlusion bodies were found to be polyhedral and to contain virions with multiple nucleocapsids. The virus was lethal to S. eridania and S. albula but not to S. frugiperda. The SperNPV-CNPSo-165 genome was 137.373 bp in size with a G + C content of 42.8%. We annotated 151 ORFs with 16 ORFs unique among baculoviruses. Phylogenetic inference indicated that this virus was closely related to the most recent common ancestor of other Spodoptera-isolated viruses.

A novel cypovirus found in a betabaculovirus co-infection context contains a poxvirus immune nuclease (poxin)-related gene.

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2',3'-cGAMP-degrading enzyme family.

Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen and one of the leading causes of nosocomial infections. Moreover, the species can cause severe infections in cystic fibrosis patients, in burnt victims and cause disease in domestic animals. The control of these infections is often difficult due to its vast repertoire of mechanisms for antibiotic resistance. Phage therapy investigation with P. aeruginosa bacteriophages has aimed mainly the control of human diseases. In the present work, we have isolated and characterized a new bacteriophage, named Pseudomonas phage BrSP1, and investigated its host range against 36 P. aeruginosa strains isolated from diseased animals and against P. aeruginosa ATCC strain 27853. RESULTS: We have isolated a Pseudomonas aeruginosa phage from sewage. We named this virus Pseudomonas phage BrSP1. Our electron microscopy analysis showed that phage BrSP1 had a long tail structure found in members of the order Caudovirales. "In vitro" biological assays demonstrated that phage BrSP1 was capable of maintaining the P. aeruginosa population at low levels for up to 12 h post-infection. However, bacterial growth resumed afterward and reached levels similar to non-treated samples at 24 h post-infection. Host range analysis showed that 51.4% of the bacterial strains investigated were susceptible to phage BrSP1 and efficiency of plating (EOP) investigation indicated that EOP values in the strains tested varied from 0.02 to 1.72. Analysis of the phage genome revealed that it was a double-stranded DNA virus with 66,189 bp, highly similar to the genomes of members of the genus Pbunavirus, a group of viruses also known as PB1-like viruses. CONCLUSION: The results of our "in vitro" bioassays and of our host range analysis suggested that Pseudomonas phage BrSP1 could be included in a phage cocktail to treat veterinary infections. Our EOP investigation confirmed that EOP values differ considerably among different bacterial strains. Comparisons of complete genome sequences indicated that phage BrSP1 is a novel species of the genus Pbunavirus. The complete genome of phage BrSP1 provides additional data that may help the broader understanding of pbunaviruses genome evolution.

Assembly of tomato blistering mosaic virus-like particles using a baculovirus expression vector system.

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.

Cell-line-dependent crystal morphology and sublocalization of the Thyrinteina arnobia cypovirus polyhedrin expressed from a recombinant baculovirus.

We describe an unexpected feature observed for the heterologous expression of the Thyrinteina arnobia cypovirus polyhedrin from a recombinant baculovirus infection in different insect cell lines. The in cellulo-formed crystals varied in size and shape depending on the cell line. Crystals formed in Trichoplusia ni-derived cells were cubic (0.1-2 µm) and localized in both the nucleus and cytoplasm, whereas those formed in Spodoptera frugiperda-derived cells were ovate and ellipsoidal (0.1-3 µm) and also localized in both the nucleus and cytoplasm. The molecular basis for differences in the morphology, size, and location of cypovirus occlusion bodies is unclear, and cellular proteins might play a role in their formation and location.

A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

A betabaculovirus encoding a gp64 homolog.

BACKGROUND: A betabaculovirus (DisaGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pests of the sugarcane and other monocot cultures in Brazil. RESULTS: The complete genome sequence of DisaGV was determined using the 454-pyrosequencing method. The genome was 98,392 bp long, which makes it the smallest lepidopteran-infecting baculovirus sequenced to date. It had a G + C content of 29.7% encoding 125 putative open reading frames (ORF). All the 37 baculovirus core genes and a set of 19 betabaculovirus-specific genes were found. A group of 13 putative genes was not found in any other baculovirus genome sequenced so far. A phylogenetic analysis indicated that DisaGV is a member of Betabaculovirus genus and that it is a sister group to a cluster formed by ChocGV, ErelGV, PiraGV isolates, ClanGV, CaLGV, CpGV, CrleGV, AdorGV, PhopGV and EpapGV. Surprisingly, we found in the DisaGV genome a G protein-coupled receptor related to lepidopteran and other insect virus genes and a gp64 homolog, which is likely a product of horizontal gene transfer from Group 1 alphabaculoviruses. CONCLUSION: DisaGV represents a distinct lineage of the genus Betabaculovirus. It is closely related to the CpGV-related group and presents the smallest genome in size so far. Remarkably, we found a homolog of gp64, which was reported solely in group 1 alphabaculovirus genomes so far.

Functional characterization of hesp018, a baculovirus-encoded serpin gene.

The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase (PO) cascade - a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge, hesp018 is the first viral serpin homologue to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited PO activation when mixed with lepidopteran haemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by fourfold in orally infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the PO response, during infection of host insects.

The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

Complete genome sequence and structural characterization of a novel iflavirus isolated from Opsiphanes invirae (Lepidoptera: Nymphalidae).

Opsiphanes invirae (Lepidopera: Nymphalidae) is a common pest of the African oil palm tree (Elaeis guineensis) in Brazil. Dead larvae were collected in canopy of oil palm trees cultivated in the amazon region (Para State) and analyzed for viral infection. Electron microscopy of caterpillar extracts showed an icosahedral picorna-like virus particle with 30nm in diameter. Total RNA extracted from partially purified virus particles was sequenced. A contig of 10,083 nucleotides (nt) was identified and showed to encode one single predicted polyprotein with 3185 amino acid residues. Phylogenetic analysis showed that the new virus was closely related to another lepidopteran infective virus Spodoptera exigua iflavirus 1(SeIV-1), with 35% amino acid pairwise identity. The novel virus fulfils all ICTV requirements for a new iflavirus species and was named Opsiphanes invirae Iflavirus 1 (OilV-1).

Genome sequence of Erinnyis ello granulovirus (ErelGV), a natural cassava hornworm pesticide and the first sequenced sphingid-infecting betabaculovirus.

BACKGROUND: Cassava (Manihot esculenta) is the basic source for dietary energy of 500 million people in the world. In Brazil, Erinnyis ello ello (Lepidoptera: Sphingidae) is a major pest of cassava crops and a bottleneck for its production. In the 1980s, a naturally occurring baculovirus was isolated from E. ello larva and successfully applied as a bio-pesticide in the field. Here, we described the structure, the complete genome sequence, and the phylogenetic relationships of the first sphingid-infecting betabaculovirus. RESULTS: The baculovirus isolated from the cassava hornworm cadavers is a betabaculovirus designated Erinnyis ello granulovirus (ErelGV). The 102,759 bp long genome has a G + C content of 38.7%. We found 130 putative ORFs coding for polypeptides of at least 50 amino acid residues. Only eight genes were found to be unique. ErelGV is closely related to ChocGV and PiraGV isolates. We did not find typical homologous regions and cathepsin and chitinase homologous genes are lacked. The presence of he65 and p43 homologous genes suggests horizontal gene transfer from Alphabaculovirus. Moreover, we found a nucleotide metabolism-related gene and two genes that could be acquired probably from Densovirus. CONCLUSIONS: The ErelGV represents a new virus species from the genus Betabaculovirus and is the closest relative of ChocGV. It contains a dUTPase-like, a he65-like, p43-like genes, which are also found in several other alpha- and betabaculovirus genomes, and two Densovirus-related genes. Importantly, recombination events between insect viruses from unrelated families and genera might drive baculovirus genomic evolution.

Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus.

Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery.

Complete genome sequence of the first non-Asian isolate of Bombyx mori nucleopolyhedrovirus.

Brazil is one of the largest silk producers in the world. The domesticated silkworm (Bombyx mori) was formally introduced into the country in the twentieth century, and the state of Paraná is the main national producer. During larval stages, B. mori can be afflicted by many different infectious diseases, which lead to substantial losses in silk production. In this work, we describe the structure and complete genome sequence of the first non-Asian isolate of Bombyx mori nucleopolyhedrovirus (BmNPV), the most important silkworm pathogen. The BmNPV-Brazilian isolate is a nucleopolyhedrovirus with singly enveloped nucleocapsids within polyhedral occlusion bodies. Its genome has 126,861 bp with a G + C content of 40.4 %. Phylogenetic analysis clustered the virus with the Japanese strain (BmNPV-T3). As expected, we have detected intra-population variability in the virus sample. Variation along homologous regions (HRs) and bro genes was observed; there were seven HRs, deletion of bro-e, and division of bro-a into two ORFs. The study of baculoviruses allows for a better understanding of virus evolution providing insight for biological control of insect pests or protection against the pernicious disease caused by these viruses.

The multispecies stinkbug iflavirus Halyomorpha halys virus detected in the multispecies stinkbug egg parasitoid microwasp, Telenomus podisi (Ashmead) (Hymenoptera: Platygastridae).

Wasps are important parasitoids of stinkbugs and frequently exposed to various types of microorganisms through environmental contact and fecal-oral transmission route. Many parasitize stinkbug eggs and are commercially used in the field to control insect population. The parasitoid T. podisi is known for its high parasitism capacity and ability to target multiple species of stinkbugs. In this study we asked whether T. podisi exposed to eggs infected by a multispecies asymptomatic stinkbug virus, the Halyomorpha halys virus (HhV) would get infected. HhV is a geographically distributed multispecies iflavirus previously found to infect four stinkbug hosts, including three Brazilian species, Chinavia ubica, Euschistus heros and Diceraeus melacanthus, and T. posidi can parasitize all of them. As results, RT-PCR screening revealed positive samples for the HhV genome in two out of four tested pools of T. podisi, whereas the antigenome, indicative of replicative activity, was not detected. The wasps were raised in E. heros eggs that presented both the genome and the antigenome forms of the HhV genome. Subsequent RNA-deep sequencing of HhV positive T. podisi RNA pools yielded a complete genome of HhV with high coverage. Phylogenetic analysis positioned the isolate HhV-Tp (isolate Telenomus podisi) alongside with the stinkbug HhV. Analysis of transcriptomes from several hymenopteran species revealed HhV-Tp reads in four species. However, the transmission mechanism and the ecological significance of HhV remain elusive, warranting further studies to illuminate both the transmission process and its capacity for environmental propagation using T. podisi as a potential vector.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

BACKGROUND: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. RESULTS: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. CONCLUSIONS: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

RPS6 transcriptional modulation in neural tissues of Nauphoeta cinerea during streptozotocin-associated sugar metabolism impairment.

The use of insects to model molecular events that characterize degenerative conditions was originally met with scepticism. However, the discovery of insect insulin-like peptides in the 1970's and the demonstration of evolutionary conservation of insulin-related signalling from insects to mammals have highlighted the importance and reduced cost of insect models in biomedical research. Here, we expand on our earlier described modelling of streptozotocin-induced brain glucose metabolic disruption in Nauphoeta cinerea, using RNA-sequencing analysis to study the transcriptional and genetic signatures of degeneration and stress signalling when glucose levels are elevated in the brain of the lobster cockroach. Nymphs were randomly divided into three groups: Control (0.8% NaCl), and two single streptozotocin injection doses (74 nmol and 740 nmol). The transcriptional analyses featured a dysregulation of 226 genes at high dose STZ treatment and 278 genes at the low dose. Our mRNA-sequencing data showed that ribosomal protein genes were the most upregulated genes at both 74 and 740 nmol STZ treatment. We therefore used RT-qPCR and relative transcriptional methods to validate our proposed mechanism of brain glucose toxicity-induced degeneration in Nauphoeta cinerea, which involved the upregulation of ribosomal proteins and rpS6 regulators (mTORC1, protein kinases, casein kinase 1 and Death-associated protein kinase), the upregulation of MAPK cascades (RAS, ERK, P38 and JNK), alongside the downregulation of the PI3K/AKT cascade. Taken together, this study highlights the remarkable opportunity for Nauphoeta cinerea use as an experimental organism in hyperglycaemia, degeneration, and stress signalling.

Exploring viral infections in honey bee colonies: insights from a metagenomic study in southern Brazil.

The decline in honey bee colonies in different parts of the world in recent years is due to different reasons, such as agricultural practices, climate changes, the use of chemical insecticides, and pests and diseases. Viral infections are one of the main causes leading to honey bee population declines, which have a major economic impact due to honey production and pollination. To investigate the presence of viruses in bees in southern Brazil, we used a metagenomic approach to sequence adults' samples of concentrated extracts from Apis mellifera collected in fifteen apiaries of six municipalities in the Rio Grande do Sul state, Brazil, between 2016 and 2017. High-throughput sequencing (HTS) of these samples resulted in the identification of eight previously known viruses (Apis rhabdovirus 1 (ARV-1), Acute bee paralysis virus (ABPV), Aphid lethal paralysis virus (ALPV), Black queen cell virus (BQCV), Bee Macula-like virus (BeeMLV), Deformed wing virus (DWV), Lake Sinai Virus NE (LSV), and Varroa destructor virus 3 (VDV-3)) and a thogotovirus isolate. This thogotovirus shares high amino acid identities in five of the six segments with Varroa orthomyxovirus 1, VOV-1 (98.36 to 99.34% identity). In contrast, segment 4, which codes for the main glycoprotein (GP), has no identity with VOV-1, as observed for the other segments, but shares an amino acid identity of 34-38% with other glycoproteins of viruses from the Orthomyxoviridae family. In addition, the putative thogotovirus GP also shows amino acid identities ranging from 33 to 41% with the major glycoprotein (GP64) of insect viruses of the Baculoviridae family. To our knowledge, this is the second report of a thogotovirus found in bees and given this information, this thogotovirus isolate was tentatively named Apis thogotovirus 1 (ATHOV-1). The detection of multiple viruses in bees is important to better understand the complex interactions between viruses and their hosts. By understanding these interactions, better strategies for managing viral infections in bees and protecting their populations can be developed.