Doreen Winstanley

Use of Whole Genome Sequence Data To Infer Baculovirus Phylogeny

ABSTRACT Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) andGranulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

The complete sequence of the Cydia pomonella granulovirus genome

The nucleotide sequence of the DNA genome of Cydia pomonella granulovirus (CpGV) was determined and analysed. The genome is composed of 123500 bp and has a G+C content of 45.2%. It contains 143 ORFs of 150 nucleotides or more that show minimal overlap. One-hundred-and-eighteen (82.5%) of these putative genes are homologous to genes previously identified in other baculoviruses. Among them, 73 are homologous to genes of Autographa californica nucleopolyhedrovirus (AcMNPV), whereas 108 and 98 are homologous to genes of Xestia c-nigrum GV (XcGV) and Plutella xylostella GV (PxGV), respectively. These homologues show on average 37.4% overall amino acid sequence identity to those from AcMNPV and 45% to those from XcGV and PxGV. The CpGV gene content was compared to that of other baculoviruses. Several genes reported to have major roles in baculovirus biology were not found in the CpGV genome, such as gp64, the major budded virus glycoprotein gene in some nucleopolyhedroviruses, and lef-7, involved in DNA replication. However, the CpGV genome encodes the large and small subunits of ribonucleotide reductase, three inhibitor of apoptosis (iap) homologues and two protein tyrosine phosphatases. The CpGV, PxGV and XcGV genomes present a noticeably high level of conservation of gene order and orientation. A striking feature of the CpGV genome is the absence of typical homologous repeat sequences. However, it contains one major repeat region and 13 copies of a single 73-77 bp imperfect palindrome.

Biocontrol of Pests of Apples and Pears in Northern and Central Europe: 1. Microbial Agents and Nematodes

The viral, bacterial, fungal and nematode pathogens of arthropod pests of apple and pear in northern and central Europe and their use as biocontrol agents are reviewed. Baculoviruses are important viral pathogens of several lepidopterous pests of apple and pear but other viral pathogens have not been investigated in depth and are little known. The granuloviruses of codling moth, Cydia pomonella (CpGV), and to a lesser extent, of the summer fruit tortrix moth, Adoxophyes orana (AoGV), have been researched extensively and are exploited as biological control agents. Commercial development and use has been limited because of their high costs, slow action, short persistence and specificity relative to broad-spectrum pesticides. The widespread development of strains of codling moth multi-resistant to insecticides and the desire to reduce dependence on pesticides have improved the commercial prospects of CpGV and use is likely to increase. The development of a genetically improved egt-strain of CpGV (lacking the...

The complete sequence of the Adoxophyes orana granulovirus genome.

The nucleotide sequence of the Adoxophyes orana granulovirus (AdorGV) DNA genome was determined and analysed. The genome contains 99,657 bp and has an A + T content of 65.5%. The analysis predicted 119 ORFs of 150 nucleotides or larger that showed minimal overlap. Of these putative genes, 104 (87%) were homologous to genes identified previously in other baculoviruses. The mean overall amino acid identity of AdorGV ORFs was highest with CpGV ORFs at 48%. Sixty-three ORFs were conserved among all lepidopteran baculoviruses and are considered to be common baculoviral genes. Several genes reported to have major roles in baculovirus biology were not found in the AdorGV genome. These included chitinase and cathepsin, which are involved in the liquefaction of the host, which explains why AdorGV-infected insects do not degrade in a typical manner. The AdorGV genome encoded two inhibitor of apoptosis (iap) genes iap-3 and iap-5. Among all of the granuloviruses genomes there was a very high level of gene collinearity. The genes shared by AdorGV and CpGV had exactly the same order along the genome with the exception of one gene, iap-3. The AdorGV genome did not contain typical homologous region (hr) sequences. However, it contained nine repetitive regions in the genome.

Replication of Cydia pomonella granulosis virus in cell cultures

Several primary cell lines that support the complete replication of Cydia pomonella granulosis virus have been established from one culture of C. pomonella embryonic cells. Virus passaged three times in cells and once in larvae showed no change in restriction enzyme fragment patterns. Stages in virus replication observed by electron microscopy resembled those from in vivo studies. Cell lines that were maintained at or below 21 degrees C retained susceptibility to virus over a period of 4 years whereas the same cell lines maintained at 27 degrees C gradually lost their susceptibility and eventually could not be infected at all.

Identification, tissue localisation and physiological effect in vitro of a neuroendocrine peptide identical to a dipteran Leu-callatostatin in the codling moth Cydia pomonella (Tortricidae: Lepidoptera)

Abstract.A neuroendocrine peptide of the Leu-callatostatin family, LPVYNFGL-NH2, has been isolated from tissue extracts of 5th instar larvae of the codling moth, Cydia pomonella (Lepidoptera). It is identical to a peptide previously isolated from the blowfly, Calliphora vomitoria (Diptera). The distribution of this peptide within the tissues of C. pomonella has been mapped by immunocytochemistry using antisera raised against LPVYNFGL-NH2. Midgut endocrine cells contain Leu-callatostatin immunoreactivity, as do several paired Leu-callatostatin neurones in the brain and ventral nerve cord. Within the visceral nervous system, the frontal ganglion contains four Leu-callatostatin neurones. Axons from these cells combine with others originating from neurones in the brain and project within the nervi cardiostomatogastrici to innervate the tissues of the foregut. In particular, the oesophageal valve has a prominent ring of Leu-callatostatin-immunoreactive fibres. The synthetic peptide, LPVYNFGL-NH2, has a potent reversible inhibitory effect in vitro on all visible forms of spontaneous contractile activity of the foregut, including closure of the oesophageal valve. Complete myoinhibition is observed at peptide concentrations from 10−10 to 10−16 M. These results, in conjunction with the results of similar studies on cockroaches, crickets and flies, suggest that the Leu-callatostatins are a ubiquitous family of insect neuroendocrine peptides with an important role in the control of gut motility.

Densovirus induces winged morphs in asexual clones of the rosy apple aphid, Dysaphis plantaginea.

Winged morphs of aphids are essential for their dispersal and survival. We discovered that the production of the winged morph in asexual clones of the rosy apple aphid, Dysaphis plantaginea, is dependent on their infection with a DNA virus, Dysaphis plantaginea densovirus (DplDNV). Virus-free clones of the rosy apple aphid, or clones infected singly with an RNA virus, rosy apple aphid virus (RAAV), did not produce the winged morph in response to crowding and poor plant quality. DplDNV infection results in a significant reduction in aphid reproduction rate, but such aphids can produce the winged morph, even at low insect density, which can fly and colonize neighboring plants. Aphids infected with DplDNV produce a proportion of virus-free aphids, which enables production of virus-free clonal lines after colonization of a new plant. Our data suggest that a mutualistic relationship exists between the rosy apple aphid and its viruses. Despite the negative impact of DplDNV on rosy apple aphid reproduction, this virus contributes to their survival by inducing wing development and promoting dispersal.

Baculovirus resistance in codling moth (Cydia pomonella L.) caused by early block of virus replication

An up to 10,000-fold resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV) was observed in field populations of codling moth, C. pomonella, in Europe. Following different experimental approaches, a modified peritrophic membrane, a modified midgut receptor, or a change of the innate immune response could be excluded as possible resistance mechanisms. When CpGV replication was traced by quantitative PCR in different tissues of susceptible and resistant insects after oral and intra-hemocoelic infection, no virus replication could be detected in any of the tissues of resistant insects, suggesting a systemic block prior to viral DNA replication. This conclusion was corroborated by fluorescence microscopy using a modified CpGV (bacCpGV(hsp-eGFP)) carrying enhanced green fluorescent gene (eGFP), which showed that infection in resistant insects did not spread. In conclusion, the different lines of evidence indicate that CpGV can enter but not replicate in the cells of resistant codling moth larvae.

The origins of replication of granuloviruses

The genomes of eight granuloviruses (GVs), have been analyzed for the presence of homologous regions (hrs) that may act as origins of replication. Thirteen 74–76-bp palindromes within 11 hrs have previously been identified in the Cydia pomonella GV (CpGV) genome and found to replicate in an infection-dependent DNA replication assay. We report a further palindrome within one of the hrs, which was found to replicate, bringing the total to 14 palindromes. We also report imperfect palindromes, with similar 13-bp end sequences to the CpGV palindromes, within the Adoxophyes orana GV, Cryptophlebia leucotreta GV (CrleGV), Choristoneura occidentalis GV and Phthorimaea operculella GV genomes. No hrs were detected in Agrotis segetum GV, and no additional hrs or palindromes, other than those published, were detected in the Plutella xylostella GV and Xestia c-nigrum GV genomes. Several putative hrs from the GVs were tested for replication in C. pomonella cells using a CpGV-dependent replication assay. Two CrleGV hrs were found to replicate at a low level.

The Comparative Susceptibility of the Diamondback Moth Plutella xylostella and Some Other Major Lepidopteran Pests of Brassica Crops to a Range of Baculoviruses

The susceptibility of larvae of the Diamondback moth (DBM), Plutella xylostella to infection by three baculoviruses was evaluated in the laboratory using a microdroplet feeding assay. The viruses tested were a granulovirus (GV), originally isolated in Taiwan from P. xylostella larvae (Px GV-Taiwan); the nucleopolyhedrovirus (NPV) from Galleria mellonella (Gm NPV), and the NPV from Autographa californica (Ac NPV). Neonate P. xylostella larvae were susceptible to infection by all three viruses. In an extensive series of bioassays carried out over a 21-month period, LD 50S for neonate DBM larvae ranged from 1.0-8.9 viral occlusion bodies (OB) for Px GV-Taiwan, and 9.5-30.2 OB for Gm NPV and Ac NPV. LT 50S for the three viruses ranged from 3.8-6.0 days at 27 C, with Gm NPV having a significantly shorter LT 50 than the other two viruses. Second and third instar larvae of P. xylostella were significantly less susceptible to infection by Px GV-Taiwan (LD 50 s ranging from 18-57 OB/larva) than were neonate larvae...