Norman E. Crook

In vivo isolation of baculovirus genotypes

Restriction endonuclease profiles of a wild-type granulosis virus from Artogeia rapae (ArGV3) and a nuclear polyhedrosis virus from Lymantria dispar (LdMNPV) indicated that the preparations were genotypically heterogeneous. Eight constituent genotypes were isolated from ArGV3 by low mortality dose infections of late instar A. rapae and analysis of progeny viral DNA recovered from individual cadavers. Three distinct genotypes were isolated from LdMNPV by the same approach, using L. dispar larvae. These results show that larval mortality can occur as a consequence of productive infection by a single virus particle. Comparative physical mapping of the eight ArGV3 genotypes suggested that their diversity may be partly attributable to recombination during natural coinfection.

Comparison of Three Methods of ELISA for Baculoviruses

Summary Direct, indirect and double antibody sandwich methods of ELISA were examined for their ability to detect and discriminate between granulosis viruses from Pieris brassicae, Agrotis segetum and Cydia pomonella and for their specificity in the presence of host material. The indirect method was the most sensitive, capable of detecting down to about 1 ng of dissolved capsules/ml compared with 10 ng/ml for the double antibody sandwich method and 25 ng/ml for the direct method. The double antibody sandwich method showed greatest discrimination between different granulosis viruses; the direct method was not quite so specific and the indirect method varied considerably depending upon the antibody concentration used although even with this latter method cross-reactions were restricted to baculo-viruses and did not occur with occluded insect viruses in other taxonomic groups. All three methods were highly specific in the presence of large amounts of host material and could be used to assay virus in diseased larvae, although at very high levels of contamination, equivalent to adding 100 healthy larvae to one diseased larva, only the double antibody sandwich method could still reliably detect virus though with reduced absorbance readings. Possible reasons why the ELISA shows much greater discrimination between different Baculovirus inclusion bodies than other serological techniques are discussed.

Variation in Cydia pomonella Granulosis Virus Isolates and Physical Maps of the DNA from Three Variants

Summary Cydia pomonella granulosis viruses (CpGV) from seven different sources in Europe, America and New Zealand were compared by restriction enzyme analysis. Most samples were indistinguishable from the Mexican isolate (CpGV-M). Isolates from Russia (CpGV-R) and England (CpGV-E) showed small genotypic differences. CpGV-E was shown to be a mixture of two variants, E1 and E2. CpGV-E1 was indistinguishable from CpGV-M. A physical map of CpGV-M was constructed for the enzymes EcoRI, BamHI, HindIII, SmaI and ApaI. A comparison of fragment profiles allowed construction of maps for CpGV-R and CpGV-E2. Relative to CpGV-M, CpGV-R had a single deletion of 2.4 kbp and CpGV-E2 was modified in one area resulting in an additional EcoRI site, a shift in a BamHI site and in total about 1 kbp more DNA. The map was orientated by locating the granulin gene using the cloned granulin gene from Trichoplusia ni GV as a probe. There was no significant difference between the infectivities of the Mexican, Russian and English isolates for neonate larvae.

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.

A comparison of the granulosis viruses from Pieris brassicae and Pieris rapae

The granulosis viruses of Pieris brassicae and P. rapae have been compared biochemically and biologically. No differences were found between virus capsules when examined by immunodiffusion, ELISA, or SDS-polyacrylamide gel electrophoresis. The virus particles were identical by immunodiffusion but distinguishable by ELISA. Comparison of virus particle polypeptides on SDS-polyacrylamide gels indicated small differences in molecular weight in three of the polypeptides of the virus envelope but no difference between the nucleocapsids. There were several differences between the DNA fragments produced by digestion with EcoRI, BamH1, and HindIII restriction endonucleases from which the homology between the two DNAs was calculated to be 97.7%. The small structural difference between the two viruses is associated with a very large difference in their virulence for P. brassicae; the LD50 with P. rapae GV being at least 1000 times greater than with P. brassicae GV. P. rapae was much more susceptible to either virus than P. brassicae and in this case the LD50 values were not significantly different for the two viruses.

Comprehensive physical map of the Cydia pomonella granulovirus genome and sequence analysis of the granulin gene region.

A cloned strain of Cydia pomonella granulovirus, CpGV-M1, was obtained using successive rounds of an in vivo limiting dilution method. A detailed physical map of the genome was constructed using 11 restriction enzymes. The region containing the granulin gene and an open reading frame immediately upstream of the granulin gene was sequenced. This region showed a high degree of homology to the equivalent region from Cryptophlebia leucotreta granulovirus with 98% amino acid identity for the granulins and 68% identity for the putative polypeptides encoded by the upstream ORFs. These latter polypeptides contained two zinc finger-like motifs and showed a low degree of homology to ME53 from Autographa californica nucleopolyhedrovirus (AcMNPV). Evidence is presented for a similar upstream ORF in Artogeia rapae GV also. Hybridization studies showed that the CpGV genome had a similar overall organization to the Artogeia rapae GV genome. Hybridization between CpGV and AcMNPV was limited to fragments spanning about 15% of each genome suggesting that very few genes are highly conserved between GVs and NPVs.

Cytoplasmic polyhedrosis virus classification by electropherotype; validation by serological analyses and agarose gel electrophoresis.

Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris-HCl/Tris-glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris-acetate-EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates.

Physical maps of the genomes of four variants of Artogeia rapae granulosis virus

Summary The genomes of four closely related variants of Artogeia rapae granulosis virus, isolated in England and New Zealand, have been mapped for the restriction endonucleases EcoRI, BamHI, HindIII, KpnI, XhoI, PstI, BglI, and SmaI by secondary digestion of isolated restriction fragments. A total of 69, 70 or 71 fragments were unambiguously mapped for these isolates. A modified system of fragment lettering was adopted so that, for each enzyme, the same letter could be used for colinear fragments from each isolate despite loss or gain of restriction sites. The sizes of the viral DNAs varied slightly, but were all about 110 kbp. The zero point for the maps was taken as the start of the granulin gene which was orientated in the same direction as the polyhedrin gene on the map of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA. Differences between the four variants are discussed in relation to their insect host range.

Restriction enzyme analysis of granulosis viruses isolated from Artogeia rapae and Pieris brassicae

Summary Thirteen isolates of granulosis virus from Artogeia (= Pieris) rapae and two from Pieris brassicae were compared by restriction enzyme analysis. All the isolates gave very similar fragment profiles with XhoI, SmaI and BglI. but at least 11 of them could be distinguished using EcoRI, BstI and HindIII. Similarities and differences between profiles suggested that the isolates could be placed in three subtypes. This subtyping correlated closely with the geographical origin of the isolates, which came from Europe, North America, Asia and Australasia. All the isolates were highly infectious for A. rapae with median lethal dose values for third instar larvae ranging from 102·3 to 102·6. Only two of four isolates of one subtype had significant infectivity for third instar P. brassicae; thus, this broader host range did not correlate with grouping by restriction enzyme analysis.