A comparison of six cypovirus isolates by cross-hybridisation of their dsRNA genome segments.

Genetic relationships between the genome segments of six cypovirus (CPV) isolates were analysed by RNA cross-hybridisation. These included three type 1 viruses and single isolates of types 2, 5 and 12, which collectively are identical to those previously compared by serology and electrophoresis [Mertens et al. (1989), J Gen Virol 70: 173-185]. Since only genome segment 10 of three cypovirus types and segments 8 and 9 of a single virus strain (of type 1) have currently been sequenced, this initial study provides some additional information on sequence variation/similarity in each of the ten genome segments. The RNA of the type 1 viruses showed high levels of cross-hybridisation. Significant but much lower levels of cross-hybridisation were detected between type 1 and the related type 12 CPV. However, only very low levels of cross-hybridisation were detected between the other pairs of viruses. Apart from evidence of a slightly higher level of sequence similarity between the largest segments, the RNA sequence appeared to vary uniformly across the whole genome. There was no evidence for any type specific RNA sequences restricted to individual genome segment(s). The sequence variation, reflected in the levels of RNA sequence similarity and cross hybridisation, correlates well with serological data, showing large differences between CPV types and supports the continued use of electropherotype as one of the 'species parameters' for the classification of cypoviruses.

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.

Properties of a novel DNA virus from the tsetse fly, Glossina pallidipes.

Virus particles were isolated from hypertrophied salivary glands of the tsetse fly, Glossina pallidipes collected near Mombasa, Kenya. Purified virus particles were rod-shaped, 57 nm wide by 700 to 1300 nm long. Particle lengths fell into two size classes, with 'short' particles averaging 869 nm and 'long' particles 1175 nm. The virus particles morphologically resembled elongated baculovirus nucleocapsids although, unlike baculoviruses, no fully enveloped virions were found in purified preparations. The particles contained double-stranded DNA which appeared to be linear when analysed by electrophoresis in agarose gels, ethidium bromide-caesium chloride gradient centrifugation or electron microscopy (EM). There was some evidence for the DNA being heterogeneous in size from EM studies and from the observation that restriction enzyme analysis failed to provide a clear profile of DNA fragments. Protein from purified virions contained at least 12 polypeptides with a major component of 39 000 mol. wt. These results suggest that the virus cannot be placed in any of the existing taxonomic groupings of DNA viruses.

Comparison of the virus-associated polymerases of cytoplasmic polyhedrosis virus types 1 and 2.

A detailed comparison was made of the virus-associated polymerase activities of cytoplasmic polyhedrosis virus (CPV) types 1 and 2 which had previously been shown to differ in their response to the methyl donor S-adenosyl-L-methionine (AdoMet). While the type 1 CPV polymerase was approximately twice as active as the type 2 CPV enzyme in the presence of AdoMet, temperature, pH and divalent cation optima of the two enzymes were similar. Both viruses synthesized in vitro single-stranded RNA copies of only one strand of the double-stranded RNA genome. In addition, each RNA segment of both viruses was transcribed in approximately equal amounts by weight. The results suggest that most features of CPV polymerase activity are highly conserved, even among CPV types which show substantial antigenic and biochemical differences.

The effects of S-adenosyl methionine (AdoMet) and its analogues on the control of transcription and translation in vitro of the mRNA products of two cytoplasmic polyhedrosis viruses.

S-Adenosyl methionine (AdoMet) and several structurally related compounds were added to in vitro systems for the synthesis of single-stranded RNA by cytoplasmic polyhedrosis virus (CPV) types 1 and 2. The effects of these compounds were examined on the level of transcription and methylation of the RNA products. Of the compounds tested, five increased the polymerase activity in both viruses, the most effective being the D- and L-stereoisomers of S-adenosyl homocysteine (AdoHcy), and the least effective, adenosine. L-AdoHcy, unlike D-AdoHcy, was also a competitive inhibitor of RNA methylation in the presence of [3H]AdoMet. The different response of both viruses to D- and L-AdoHcy suggests that CPV virions contain at least two functionally distinct sites to which AdoMet, or its analogues, bind. One of these is the transcription control site, while the other is the active site(s) for RNA methylation. CPV RNA synthesised in the presence of the methyl donor AdoMet was more efficiently translated in vitro in a wheat-germ translation system than RNA synthesised in the presence of methylation inhibitors. Type 2 CPV-RNA transcripts had a greater degree of methylation than type 1 CPV transcripts and were more effective in stimulating protein synthesis in the translation system. It seems likely that the allosteric control of CPV polymerase by AdoMet and its analogues, and the methylation of the transcripts, ensures the effective transcription and translation of the CPV genome and the stability of the viral messenger RNA.

A biochemical and biological comparison of three European isolates of nuclear polyhedrosis viruses from Agrotis segetum.

Isolates of multiply-enveloped nuclear polyhedrosis viruses from Agrotis segetum (Lepidoptera: Noctuidae) populations in England (AsNPVE), France (AsNPVF) and Poland (AsNPVP), were compared biochemically and for their infectivity to A. segetum and Mamestra brassicae larvae. The electrophoretic profiles of DNA restriction endonuclease fragments and viral proteins appeared identical for AsNPVE and AsNPVF. AsNPVP was distinct by these techniques, although some of the virus particle polypeptides had the same mobilities as those of the other isolates. A serological comparison of the A. segetum NPV isolates with other baculoviruses, using indirect enzyme-linked immunosorbent assay, suggested that AsNPVP was no more closely related than M. brassicae NPV to the other A. segetum NPV isolates. AsNPVP had significantly lower infectivity for neonate A. segetum larvae (LD50 = 350 inclusion bodies) than AsNPVE (10 inclusion bodies) or AsNPVF (23 inclusion bodies). AsNPVE and AsNPVF did not appear to replicate in M. brassicae larvae, while AsNPVP produced a limited infection in this species.

S-adenosyl-L-homocysteine as a stimulator of viral RNA synthesis by two distinct cytoplasmic polyhedrosis viruses.

An in vitro RNA-synthesizing system was used to study the effects of S-adenosyl-L-homocysteine, S-adenosyl-L-methionine, and adenosine on the methylation and synthesis of single-stranded RNA by two different cytoplasmic polyhedrosis viruses.

Alkaline protease associated with virus particles of a nuclear polyhedrosis virus: assay, purification, and properties.

Proteolytic activity was detected within polyhedra of the nuclear polyhedrosis virus of Spodoptera littoralis. The enzyme activity was detected by its ability to degrade the major structural polypeptide of polyhedra (polyhedrin). A quantitative assessment of activity was made by a radioassay technique using (3)H-labeled polyhedrin as the substrate. Of the structural components of polyhedra, virus particles showed the greatest specific proteolytic activity. Preparations of purified nucleocapsids were inactive. The virus particle enzyme displayed a temperature optimum for proteolysis of 30 to 40 degrees C and a pH optimum of 9.6. Its activity was inhibited by H(2+) and Cu(2+), but not by 2-mercaptoethanol. The enzyme was purified from detergent-treated virus particles by affinity column chromatography, using polyhedrin linked to cyanogen bromide-activated Sepharose. Three major envelope polypeptides (L107, L85, and L71) bound to the column at 4 degrees C, but after incubation at 31 degrees C, polypeptide L71 alone was eluted. The fractions containing this protein exhibited a specific enzyme activity more than 80-fold greater than that present in polyhedra. The possible significance of the alkaline protease, and other proteins with affinity for polyhedrin, is discussed.

The properties of two recent isolates of cytoplasmic polyhedrosis viruses.

Cytoplasmic polyhedrosis viruses (CPVs) were isolated from laboratory cultures of Bombyx mori and Spodoptera exempta. The electrophoretic profile of the RNA segments of both viruses showed similarities with that of type 1 CPV. However, whereas the virus isolate from B. mori appeared identical to type 1 CPV (as assessed by serology, as well as the comparative mobilities of RNA segments and structural polypeptides), the isolate from S. exempta was distinct. Three of the ten viral RNA segments did not co-run with type 1 RNA, and the structural polypeptides of polyhedra had different molecular weights. It is proposed that the S. exempta isolate be included as a new 'type' (type 12) in a classification of these viruses.

A provisional classification of cytoplasmic polyhedrosis viruses based on the sizes of the RNA genome segments.

The RNA genome segments of thirty-three isolates of cytoplasmic polyhedrosis viruses (CPVs) were examined by polyacrylamide gel electrophoresis. Major differences were observed in the gel profiles of the RNA segments from many of the viruses; differences which were reinforced by polyacrylamide gel electrophoresis of the virus structural proteins. As a result of these studies, a provisional classification scheme for CPVs is proposed, where viruses with similar RNA gel profiles are included within the same 'type', while isolates differing in the molecular weights of most, or all of the RNA segments are assigned to different types. Using this system, eleven distinct CPV types were recognized. All eleven CPV types, like reoviruses, probably contain ten segments of RNA with a toatl mol. wt. of approx. 15 X 10(6).

Antibodies ot double-stranded RNA: specificity and serum nucleases.

An antiserum was prepared in rabbits to the synthetic double-stranded ribonucleic acid (ds RNA) poly rI:rC. Using a liquid-phase radioimmunoassay, the antiserum cross-reacted with a natural ds RNA isolated from the cytoplasmic polyhedrosis virus of the silkworm, binding 95% of the RNA at a 1 : 20 serum dilution. Preliminary tests of the specificity of the antiserum showed that it did not bind single-stranded RNA (ss RNA) or deoxyribonucleic acid (DNA), but also revealed that the serum contained an enzyme activity which degraded ss RNA into acid-insoluble fragments. It was therefore possible that the failure to bind ss RNA resulted from the degradation of the antigen rather than from an absence of cross-reacting antibodies. However, when the serum ribonuclease activity was inhibited by macaloid, the antiserum still did not bind the ss RNA antigen. This demonstrated that the antibodies to ds RNA did not cross-react with ss RNA. The existence of serum enzymes capable of degrading nucleic acid antigens emphasizes the need for caution in assessing the specificity of such antisera.