In JoVE (1)

Other Publications (21)

Articles by Robert I. Graham in JoVE

 JoVE Immunology and Infection

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 3Crop and Environment Sciences, Harper Adams University


JoVE 55534

Other articles by Robert I. Graham on PubMed

Comparative Analgesia, Cardiovascular and Renal Effects of Celecoxib, Rofecoxib and Acetaminophen (paracetamol)

Current Pharmaceutical Design. 2002  |  Pubmed ID: 11945151

Comparisons are made between the specific COX-2 inhibitors, celecoxib and rofecoxib, and acetaminophen. The specific COX-2 inhibitors are a significant advance in therapy because their anti-inflammatory, analgesic and antipyretic activities are associated with a high degree of gastrointestinal safety. Acetaminophen is often not considered to be a potent inhibitor of COX-2 but it is a potent inhibitor of prostaglandin synthesis in intact cells after stimulation by cytokines. Its weak activity on the pathway of prostanoid synthesis involving COX-1 is shown by its weak anti-platelet activity and good gastrointestinal safety. The specific COX-2 inhibitors and acetaminophen are analgesic after dental surgery, orthopedic surgery and in osteoarthritis although acetaminophen appears to be a slightly weaker agent. The apparent analgesic activity of both the COX-2 inhibitors and acetaminophen may, in part, be due to their anti-inflammatory properties. Both groups of drugs also decrease the urinary excretion of prostacyclin metabolites consistent with inhibition of the systemic and renal activity of the COX-2 system. During repeated dosage with the specific COX-2 inhibitors, the 24 hour urinary excretion of sodium is only inhibited for the first day of treatment while the excretion of sodium is still decreased over the first 3 hours after the individual doses. Therapeutic doses of the COX-2 inhibitors and overdoses of acetaminophen have been associated with the development of occasional cases of acute renal failure. Acetaminophen also may decrease the excretion of sodium and the reason for its greater renal safety at therapeutic doses is unclear. Myocardial infarction has also been attributed to the specific COX-2 inhibitors from meta-analysis of large scale clinical trials and examination of reports of adverse drug reactions although this is still a topic of considerable discussion. No such associations have been made with acetaminophen, possibly because it is a weak inhibitor of COX-1 in platelets.

Genetically Variable Nucleopolyhedroviruses Isolated from Spatially Separate Populations of the Winter Moth Operophtera Brumata (Lepidoptera: Geometridae) in Orkney

Journal of Invertebrate Pathology. Sep, 2004  |  Pubmed ID: 15491596

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.

Detection and Characterisation of Three Novel Species of Reovirus (Reoviridae), Isolated from Geographically Separate Populations of the Winter Moth Operophtera Brumata (Lepidoptera: Geometridae) on Orkney

Journal of Invertebrate Pathology. Feb, 2006  |  Pubmed ID: 16413573

Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles in order to investigate the prevalence of virus pathogens. Reoviruses were isolated in 11 of the 13 winter moth populations sampled, with 3 novel species being detected. Two species of Cypoviridae (CPV) were isolated, Operophtera brumata CPV18 and O. brumata CPV19, with one host population suffering 46% infection prevalence of OpbuCPV19. A third virus, O. brumata Reovirus (OpbuRV), was isolated from both winter moth and a hymenopteran parasitoid wasp, Phobocampe tempestiva, which is abundant in these populations. This was identified as a non-occluded reovirus, which was clearly able to infect and persist in both the lepidopteran and the hymenopteran host. The genomes of the three viruses were characterised using gel electrophoresis and the virus structure was investigated using transmission electron microscopy. The relationship of these viruses with a baculovirus that also infects winter moth, OpbuNPV, was investigated, as well as the association of OpbuRV with P. tempestiva. The detection of such viruses is discussed with reference to studies of similar viruses in other lepidopteran and hymenopteran host systems.

Characterisation and Partial Sequence Analysis of Two Novel Cypoviruses Isolated from the Winter Moth Operophtera Brumata (Lepidoptera: Geometridae)

Virus Genes. Oct, 2007  |  Pubmed ID: 17546493

The complete nucleotide sequences of genomic segments S5 to S10 from Operophtera brumata cypovirus 18 (OpbuCPV18), and the complete nucleotide sequences of genomic segments S2, S5, S9 and S10 from Operophtera brumata cypovirus 19 (OpbuCPV19) have been determined. Each genome segment contained a single open reading frame (ORF). Conserved motifs 5' (AGUAAA....GUUAGCU) 3' were found at the ends of each segment of OpbuCPV18, whilst conserved motifs 5' (AACAAA....UUUGC) 3' were found at each segment terminus of OpbuCPV19. The putative proteins were compared with those of other members of the Reoviridae family. Phylogenetic analysis using the polyhedrin gene (S10) indicated that OpbuCPV18 was most closely related to Dendrolimus punctatus cypovirus 1, whilst OpbuCPV19 was most closely related to Trichoplusia ni cypovirus 15. In addition, analysis of S2, which encoded a putative RNA-dependant RNA polymerase gene, confirmed OpbuCPV19 belonged to the genus Cypovirus. Following the expression of the ORF from OpbuCPV19 S10, using a baculovirus expression vector, occlusion bodies were observed in insect cell culture. This demonstrated that segment 10 coded for the polyhedrin gene, capable of forming a polyhedral crystalline matrix.

Sequence Analysis of a Reovirus Isolated from the Winter Moth Operophtera Brumata (Lepidoptera: Geometridae) and Its Parasitoid Wasp Phobocampe Tempestiva (Hymenoptera: Ichneumonidae)

Virus Research. Jul, 2008  |  Pubmed ID: 18405997

A reovirus was isolated from Operophtera brumata (ObRV) and its parasitoid wasp Phobocampe tempestiva. Each of the 10 dsRNA genome segments of ObRV was sequenced and shown to contain a single open reading frame (ORF). Conserved motifs ([+ve] 5'-AAATAAA ...(G)/(T)AGGTT-3') were found at the termini of each segment, with the exception of Seg-6 and Seg-8, where the 5' termini were 5'-AACAAA...-3'. The putative proteins encoded by each segment were compared with those of other members of the family Reoviridae. Phylogenetic comparisons to published sequences for the RNA-dependent RNA polymerase genes from other reoviruses indicated that ObRV is most closely related to members of the genus Cypovirus. However, unlike the cypoviruses, ObRV has a double-layered capsid structure. When the protein encoded by ObRV Seg-10 was expressed (by inserting the open reading frame into a baculovirus expression vector) no 'occlusion bodies' were observed in the recombinant baculovirus infected insect cell cultures. This suggests that unlike the cypoviruses, Seg-10 of ObRV does not contain a polyhedrin gene. Further phylogenetic comparisons also identified relationships between Seg-2 and Seg-10 of ObRV, and genes of Diadromus pulchellus Idnoreovirus 1 (DpIRV1), suggesting that ObRV represents a new species from the genus Idnoreovirus.

Molecular Characterisation of a Cypovirus Isolated from the Western Spruce Budworm Choristoneura Occidentalis

Archives of Virology. 2008  |  Pubmed ID: 18695934

A novel cypovirus, assigned CoCPV, was isolated from natural populations of the western spruce budworm, Choristoneura occidentalis. The complete nucleotide sequences of genomic segments S2-S5 and S7-S10 were determined. Each segment contained a single open reading frame. Conserved motifs 5' (AGUUU......UUUGUGC) 3' were found at the ends of each segment. Analysis of S2, which encoded a putative RNA-dependent RNA polymerase protein, confirmed CoCPV belonged to the genus Cypovirus within the family Reoviridae. Further phylogenetic analysis using S10 (the polyhedrin gene) aligned this virus with species type-16, closely related to a cypovirus isolated from C. fumiferana.

An Intracellular Symbiont and Other Microbiota Associated with Field-collected Populations of Sawflies (Hymenoptera: Symphyta)

Canadian Journal of Microbiology. Sep, 2008  |  Pubmed ID: 18772939

Six species of sawfly (Hymenoptera: Symphyta) from four taxonomic families (Agridae, Diprionidae, Pamphiliidae, and Tenthredinidae) were collected from locations across Canada and surveyed for their associated microbiota. Total DNA was extracted from individual insects, and polymerase chain reaction (PCR) was used to amplify the conserved 16S rRNA gene from microbiota. Denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP) were undertaken to separate bacterial clones associated with the host insect. Sequencing of the PCR-DGGE and PCR--RFLP products revealed a dominance of alpha- and gamma-Proteobacteria, with most sequences showing high similarity to bacteria previously identified from other insect species and environmental samples. Additionally, a strain of the bacterial endosymbiont Wolbachia and a Wolbachia bacteriophage were identified from the mountain ash sawfly (Pristiphora geniculata).

Characterisation of a Nucleopolyhedrovirus and Spiroplasma Sp. Bacterium Associated with Outbreaking Populations of the Antler Moth Cerapteryx Graminis

Journal of Invertebrate Pathology. May, 2011  |  Pubmed ID: 21281646

A broad survey was undertaken to characterise microbes associated with larval outbreaks of the Antler moth Cerapteryx graminis in Cumbria, United Kingdom. A nucleopolyhedrovirus present in all sampled populations at ≤5% prevalence, was characterised via restriction fragment length polymorphism and partial sequencing the Polyhedrin, Lef-8 and Lef-9 genes; indicating a previously uncharacterised species most closely related to Agrotis ipsilon NPV. A survey of the host-associated bacterial community detected a species phylogenetically related to Spiroplasma sp., a male-killing phenotype previously isolated from Lepidoptera and Coleoptera, present at <63% prevalence in larvae. The implications of these associated microbes for host population dynamics are discussed.

Complete Sequence, Analysis and Organization of the Orgyia Leucostigma Nucleopolyhedrovirus Genome

Viruses. Nov, 2011  |  Pubmed ID: 22163346

The complete genome of the Orgyia leucostigma nucleopolyhedrovirus (OrleNPV) isolated from the whitemarked tussock moth (Orgyia leucostigma, Lymantridae: Lepidoptera) was sequenced, analyzed, and compared to other baculovirus genomes. The size of the OrleNPV genome was 156,179 base pairs (bp) and had a G+C content of 39%. The genome encoded 135 putative open reading frames (ORFs), which occupied 79% of the entire genome sequence. Three inhibitor of apoptosis (ORFs 16, 43 and 63), and five baculovirus repeated ORFs (bro-a through bro-e) were interspersed in the OrleNPV genome. In addition to six direct repeat (drs), a common feature shared among most baculoviruses, OrleNPV genome contained three homologous regions (hrs) that are located in the latter half of the genome. The presence of an F-protein homologue and the results from phylogenetic analyses placed OrleNPV in the genus Alphabaculovirus, group II. Overall, OrleNPV appears to be most closely related to group II alphabaculoviruses Ectropis obliqua (EcobNPV), Apocheima cinerarium (ApciNPV), Euproctis pseudoconspersa (EupsNPV), and Clanis bilineata (ClbiNPV).

Wolbachia in a Major African Crop Pest Increases Susceptibility to Viral Disease Rather Than Protects

Ecology Letters. Sep, 2012  |  Pubmed ID: 22731846

Wolbachia are common vertically transmitted endosymbiotic bacteria found in < 70% of insect species. They have generated considerable recent interest due to the capacity of some strains to protect their insect hosts against viruses and the potential for this to reduce vector competence of a range of human diseases, including dengue. In contrast, here we provide data from field populations of a major crop pest, African armyworm (Spodoptera exempta), which show that the prevalence and intensity of infection with a nucleopolydrovirus (SpexNPV) is positively associated with infection with three strains of Wolbachia. We also use laboratory bioassays to demonstrate that infection with one of these strains, a male-killer, increases host mortality due to SpexNPV by 6-14 times. These findings suggest that rather than protecting their lepidopteran host from viral infection, Wolbachia instead make them more susceptible. This finding potentially has implications for the biological control of other insect crop pests.

Male-killing Wolbachia and Mitochondrial Selective Sweep in a Migratory African Insect

BMC Evolutionary Biology. Oct, 2012  |  Pubmed ID: 23061984

Numerous recent studies have shown that resident symbiotic microorganisms of insects play a fundamental role in host ecology and evolution. The lepidopteran pest, African armyworm (Spodoptera exempta), is a highly migratory and destructive species found throughout sub-Saharan Africa, that can experience eruptive outbreaks within the space of a single generation, making predicting population dynamics and pest control forecasting extremely difficult. Three strains of Wolbachia have recently been identified infecting this species in populations sampled from Tanzania. In this study, we examined the interaction between Wolbachia pipiensis infections and the co-inherited marker, mtDNA, within populations of armyworm, as a means to investigate the population biology and evolutionary history of Wolbachia and its host.

Pest Control: Biopesticides' Potential

Science (New York, N.Y.). Nov, 2013  |  Pubmed ID: 24233706

Locusts Increase Carbohydrate Consumption to Protect Against a Fungal Biopesticide

Journal of Insect Physiology. Oct, 2014  |  Pubmed ID: 24862155

There is growing evidence to suggest that hosts can alter their dietary intake to recoup the specific resources involved in mounting effective resistance against parasites and pathogens. We examined macronutrient ingestion and disease-resistance in the Australian plague locust (Chortoicetes terminifera), challenged with a fungal pathogen (Metarhizium acridum) under dietary regimes varying in their relative amounts of protein and digestible carbohydrate. Dietary protein influenced constitutive immune function to a greater extent than did carbohydrate, indicating higher protein costs of mounting an immune defence than carbohydrate or overall energy costs. However, it appears that increased immune function, as a result of greater protein ingestion, was not sufficient to protect locusts from fungal disease. We found that locusts restricted to diets high in protein (P) and low in carbohydrate (C) were more likely to die of a fungal infection than those restricted to diets with a low P:C ratio. We hypothesise that the fungus is more efficient at exploiting protein in the insect's haemolymph than the host is at producing immune effectors, tipping the balance in favour of the pathogen on high-protein diets. When allowed free-choice, survivors of a fungus-challenge chose a less-protein-rich diet than those succumbing to infection and those not challenged with fungus locusts. These results are contrary to previous studies on caterpillars in the genus Spodoptera challenged with bacterial and baculoviral pathogens, indicating that nutrient ingestion and pathogen resistance may be a complex interaction specific to different host species and disease agents.

Densovirus is a Mutualistic Symbiont of a Global Crop Pest (Helicoverpa Armigera) and Protects Against a Baculovirus and Bt Biopesticide

PLoS Pathogens. Oct, 2014  |  Pubmed ID: 25357125

Mutualistic associations between symbiotic bacteria and their hosts are common within insect systems. However, viruses are often considered as pathogens even though some have been reported to be beneficial to their hosts. Herein, we report a novel densovirus, Helicoverpa armigera densovirus-1 (HaDNV-1) that appears to be beneficial to its host. HaDNV-1 was found to be widespread in wild populations of H. armigera adults (>67% prevalence between 2008 and 2012). In wild larval populations, there was a clear negative interaction between HaDNV-1 and H. armigera nucleopolyhedrovirus (HaNPV), a baculovirus that is widely used as a biopesticide. Laboratory bioassays revealed that larvae hosting HaDNV-1 had significantly enhanced resistance to HaNPV (and lower viral loads), and that resistance to Bacillus thuringiensis (Bt) toxin was also higher at low doses. Laboratory assays indicated that the virus was mainly distributed in the fat body, and could be both horizontally- and vertically-transmitted, though the former occurred only at large challenge doses. Densovirus-positive individuals developed more quickly and had higher fecundity than uninfected insects. We found no evidence for a negative effect of HaDNV-1 infection on H. armigera fitness-related traits, strongly suggesting a mutualistic interaction between the cotton bollworm and its densovirus.

Transgenerational Effects Modulate Density-dependent Prophylactic Resistance to Viral Infection in a Lepidopteran Pest

Biology Letters. Mar, 2015  |  Pubmed ID: 25808002

There is an increasing appreciation of the importance of transgenerational effects on offspring fitness, including in relation to immune function and disease resistance. Here, we assess the impact of parental rearing density on offspring resistance to viral challenge in an insect species expressing density-dependent prophylaxis (DDP); i.e. the adaptive increase in resistance or tolerance to pathogen infection in response to crowding. We quantified survival rates in larvae of the cotton leafworm (Spodoptera littoralis) from either gregarious- or solitary-reared parents following challenge with the baculovirus S. littoralis nucleopolyhedrovirus. Larvae from both the parental and offspring generations exhibited DDP, with gregarious-reared larvae having higher survival rates post-challenge than solitary-reared larvae. Within each of these categories, however, survival following infection was lower in those larvae from gregarious-reared parents than those from solitary-reared, consistent with a transgenerational cost of DDP immune upregulation. This observation demonstrates that crowding influences lepidopteran disease resistance over multiple generations, with potential implications for the dynamics of host-pathogen interactions.

Development of a Real-Time QPCR Assay for Quantification of Covert Baculovirus Infections in a Major African Crop Pest

Insects. Aug, 2015  |  Pubmed ID: 26463414

Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR) procedure using a 5' nuclease hydrolysis (TaqMan) probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV). The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics.

Severe GHB Withdrawal Delirium Managed with Dexmedetomidine

The Medical Journal of Australia. Sep, 2016  |  Pubmed ID: 27627929

Functional Opsin Retrogene in Nocturnal Moth

Mobile DNA. 2016  |  Pubmed ID: 27777631

Retrotransposed genes are different to other types of genes as they originate from a processed mRNA and are then inserted back into the genome. For a long time, the contribution of this mechanism to the origin of new genes, and hence to the evolutionary process, has been questioned as retrogenes usually lose their regulatory sequences upon insertion and generally decay into pseudogenes. In recent years, there is growing evidence, notably in mammals, that retrotransposition is an important process driving the origin of new genes, but the evidence in insects remains largely restricted to a few model species.

Characterization of a Novel Member of Genus Iflavirus in Helicoverpa Armigera

Journal of Invertebrate Pathology. Mar, 2017  |  Pubmed ID: 28163012

The cotton bollworm, Helicoverpa armigera, is one of the most important agricultural pests of many economic crops worldwide. Herein, we found a novel single-strand RNA virus by RNA-Seq and Polymerase Chain Reaction (PCR) method in H. armigera named Helicoverpa armigera iflavirus (HaIV), which possessed a genome with 10,017 nucleotides in length and contained a single large open reading frame (ORF) encoding a putative polyprotein of 3021 amino acids with a predicted molecular mass of 344.16kDa and a theoretical isoelectric point (pI) of 6.45. The deduced amino acid sequence showed highest similarity (61.0%) with the protein of Lymantria dispar Iflavirus 1. Phylogenetic analysis with putative RdRp amino acid sequences indicated that the virus clustered with members of the genus Iflavirus. The virus was mainly distributed in the fat body of its host and was found to be capable of both horizontal and vertical transmission. The efficiency of perorally horizontal transmission was dose dependent (100% infection rate with a viral dose of 10(8)copies/μl) while vertical transmission efficiency was found to be relatively low (<28.57%). These results suggest that we have found a novel member of genus Iflavirus in H. armigera.

Structure and Transcription of the Helicoverpa Armigera Densovirus (HaDV2) Genome and Its Expression Strategy in LD652 Cells

Virology Journal. Feb, 2017  |  Pubmed ID: 28173863

Densoviruses (DVs) are highly pathogenic to their hosts. However, we previously reported a mutualistic DV (HaDV2). Very little was known about the characteristics of this virus, so herein we undertook a series of experiments to explore the molecular biology of HaDV2 further.

Structural Proteins of Helicoverpa Armigera Densovirus 2 Enhance Transcription of Viral Genes Through Transactivation

Archives of Virology. Feb, 2017  |  Pubmed ID: 28210815

Herein, we report the identification of putative promoters for the non-structural proteins (NS) and capsid structural proteins (VP) of Helicoverpa armigera densovirus (HaDV2) as well as a potential mechanism for how these promoters might be regulated. For the first time, we report that VP is able to transactivate the VP promoter and, to a lesser degree, the NS promoter in densoviruses. In addition to this, another promoter-like sequence designated P2, when co-transfected with the VP gene, enhanced luciferase activity by approximately 35 times compared to a control. This suggests that there are two promoters for VP in HaDV2 and that the VP of parvoviruses might play a more important role in viral transcription than previously appreciated.

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