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.

Solid phase radioimmunoassay using labelled antibodies: a conceptual framework for designing assays.

A simple theoretical model for the antigen-antibody reaction is presented ans used to evaluate the optimum conditions for designing solid phase radioimmunoassay (RIA) using labelled antibodies. Both theoretical and experimental data are presented, using a wide variety of antigens and their corresponding antibodies. The types of RIA is described include the direct, the indirect, sandwich assays for detecting either antigen or antibody. The experimental results confirm in a semiquantitative manner that the greatest sensitivity of the RIA is achieved when the smallest amount of labelled antibody is used, that whenever possible the antigen/antibody ratio should be greater than unity (greater than 1), and that the formation of the antigen-antibody comples is dependent on the mass action effect.

RNA transcript mapping of the Wiseana iridescent virus genome.

The replication of Wiseana iridescent virus (WIV) was studied in Lymantria dispar tissue culture cells. Using a combination of [35S]methionine pulse-labeling and Northern blotting with WIV DNA probes, a transcriptional map of the genome was constructed. WIV has a wide dispersal of immediate-early genes with seven different regions identified. WIV has been reported to have extensive repetitive DNA sequences but no early transcription was observed in these regions. Although fine-mapping is required, some early regions (Bam L and Eco O) have been identified which are transcriptionally active at 6- and 12-h but are shut down by 24 h. These regions could provide probes for early genes and the hypothesized switch from nuclear to cytoplasmic replication for iridoviruses.

Comparison of the major capsid protein genes, terminal redundancies, and DNA-DNA homologies of two New Zealand iridoviruses.

Molecular comparisons were carried out on two iridoviruses isolated from endemic sympatric New Zealand pasture pests. These viruses, Costelytra zealandica iridescent virus (CzIV/IV16) and Wiseana iridescent virus (WIV/IV9), belong to the same virus genus but it is not known how related they are. The major capsid protein (MCP) gene from each virus was located, sequenced, and compared to the homologous gene from other iridoviruses. The MCP genes of WIV and CzIV were similar to each other (87.9% amino acid similarity) and to other iridovirus MCP genes. The MCP genes of both WIV and CzIV were most homologous to the MCP gene from Tipula iridescent virus (TIV/IV1), with amino acid similarities of 92.3 and 88.3% respectively. The genomes of WIV and CzIV were compared to other invertebrate iridoviruses using solution DNA-DNA hybridisations. Even after reducing the annealing stringency conditions hybridisation ratios never exceeded 10% indicating there is little sequence conservation between iridovirus genomes. Estimates of the size of terminal redundancies were also calculated for these viruses using pulsed-field agarose gel electrophoresis. These values ranged from 0 to 8%. These studies indicate that WIV and CzIV have distinct genomes and that the genus Iridovirus is comprised of a group of genetically diverse viruses.

Comparison of a DNA-DNA dot-blot hybridisation assay with light microscopy and radioimmunoassay for the detection of a nuclear polyhedrosis virus.

A dot-blot hybridisation assay was developed for the detection of a nuclear polyhedrosis virus (NPV) and was compared to light microscopy and radioimmunoassay (RIA). Using cloned NPV DNA labelled with 32P as a probe, a number of hybridisation assay procedures was examined. The assay was found to be more sensitive than differential staining, phase-contrast microscopy, or indirect solid-phase RIA with as few as 20 occlusion bodies (150 pg DNA) being detected. Samples do not require prior purification or DNA extraction. The assay was shown to be specific for NPV and has the potential to detect and discriminate between strains of the virus. With little modification the assay may be used to detect other insect viruses.

Rotavirus infection in New Zealand.

Rotavirus infection is commonly found in young infants admitted to hospital with gastroenteritis. An enzyme-linked immunosorbent assay (ELISA) for virus diagnosis is described and the results of testing stool specimens from 497 children with gastroenteritis, 192 neonates and 247 asymptomatic six month old infants are presented. Rotavirus infection was found in 45 percent of all children with gastroenteritis but only in 4.7 percent of neonates and 2 percent of asymptomatic infants. These results do not support the proposal that children in our community have a high incidence of subclinical infections.

Rotavirus infection in Otago: a serological study.

A method for measuring rotavirus antibody in human sera has been established using enzyme-linked immunosorbent assay (ELISA). A Simian strain of rotavirus (SA11) was used as the antigen. Serum eluted from dried blood spots on good quality chromatography paper was found suitable for analysis. Paired serum samples from children with gastroenteritis have shown a brisk antibody response in association with the presence of rotavirus in the faeces. Community studies indicate that although all older children and adults tested have detectable antibodies to rotavirus, there is a significant rise in the number of individuals with high titre antibody in the child bearing age group, after which the levels diminish. This finding suggests that repeated infections occur throughout childhood and early adult life.

Comparison of Pulsed-Field Gel Electrophoresis DNA Fingerprints of Field Isolates of the Entomopathogen Bacillus popilliae.

Eight strains of the entomopathogen Bacillus popilliae were analyzed by pulsed-field gel electrophoresis, and genomic size estimates of ;sim2,600 to 3,500 kb were obtained. The type strain, ATCC 14706, had a genomic size of 3,395 kb. For the six New Zealand isolates, the degree of similarity in the pulsed-field gel electrophoresis fingerprints may correlate with the geographical closeness of the sites of isolation. The plasmid profiles of the New Zealand isolates were also compared; four of the six strains carry plasmids in the 3.6- to 9.7-kb size range.

Sequence comparison of the major capsid protein gene from 18 diverse iridoviruses.

Insect iridoviruses (IV) have been found on all continents of the world and in a broad range of insect hosts. The host range for a single strain can cross several insect orders. This along with a paucity of molecular information on all but a few members has led to confusion in the taxonomy and classification of these viruses and in the identification of potentially novel isolates. To address this problem consensus PCR primers were designed to amplify and sequence a 500 bp region of the major capsid protein (MCP) gene. PCR products were amplified from eighteen IVs belonging to the genus Iridovirus. No product was observed for the chloriridovirus IV3. Phylogenetic analysis of the partial MCP gene sequence showed that the iridovirus genus can be divided into three groups. These results support previous studies where a range of molecular techniques were used. Group I contained PjIV and IV31, group II contained IV6 (CIV), IV21, and IV28, and group III contained IV1 (TIV), IV2 (SIV), IV9 (WIV), IV10, IV16, (CzIV), IV18, IV22, IV23 (BbIV), IV24, IV29, IV30, AgIV and an undescribed weevil IV. There was no correlation of relatedness with host of isolation but there was some correlation with geographic region of isolation. Sequence analysis also raised issues concerning the purity of some virus stocks and supported the view that some isolates should be considered as variants of one virus species.

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.

Effect of alkaline protease on the antigenic nature of wiseana nuclear polyhedrosis virus polyhedron protein.

Polyhedron protein from Wiseana spp. nuclear polyhedrosis virus was found to be degraded by an alkali protease when polyhedra are dissolved in alkali. The protease activity did not occur at high pH (0.1 M NaOH) and was inactivated by heating polyhedra to 70 degrees C for 3 h. The products from the protease degradation of Wiseana spp. nuclear polyhedrosis virus polyhedron protein retain the antigenicity of undegraded polyhedron protein as measured by the direct solid-phase radioimmunoassay and immunoadsorption. Degradation products below 27,000 daltons could not be detected by the sandwich radioimmunoassay, indicating that they are probably monovalent.

Induction of viral interference: effects of poly rI-rC and diethylaminoethyl-dextran on the activity of the antiviral protein.

The activity of the antiviral protein induced by various ratios of poly rI.rC and diethylaminoethyl (DEAE)-dextran was studied. It was found that, when large doses of poly rI.rC were used, very little viral interference was observed. This effect was initially attributed to the cells being refractory for production of antiviral protein. Subsequent experiments offered alternative explanations suggesting that, at any given dosage of poly rI.rC, an excess of DEAE-dextran is necessary for the production of viral interference. It is suggested that DEAE-dextran acts by exposing a cell receptor site for poly rI.rC.

Labeling of antibodies with 3H-acetate.

Antibodies can be labeled with (3)H-iodoacetate at pH 9.8 without significant loss of biological activity, and the labeled antibody can be used to detect viral antigens.

Physical mapping of the DNA genome of insect iridescent virus type 9 from Wiseana spp. larvae.

A physical map for the DNA genome of insect iridescent virus type 9 isolated from Wiseana spp. larvae [Lepidoptera: Hepialidae] was constructed using the restriction enzymes BamHl, EcoRl, and Pstl. Viral DNA was cloned into the plasmids pBR328 and pUC8 using Escherichia coli strains HB101 and JM83, respectively. The physical map for BamHl, EcoRl, and Pstl was constructed by multiple enzyme analysis and Southern hybridization of cloned viral DNA. Statistical analysis of restriction data by computer-aided linear modeling supported the physical map produced and indicated a total genome size of 192.5 kb. Due to the cyclic permutation of iridescent virus genomes the map is presented in a circular form.

Physicochemical properties of tipula iridescent virus.

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 x 10(8), with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 +/- 0.2%. At 260 mmu, the virus gave an uncorrected absorbance of 18.2 cm(2)/mg of virus and a light-scattering corrected absorbance of 9.8 cm(2)/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.

Detection of double-stranded RNA in Semliki Forest virus-infected cells by an indirect solid phase radioimmunoassay: an assay for interferon.

An indirect solid phase radioimmunoassay using rabbit antibodies to the synthetic double-stranded RNA poly rI-rC and 125I-labelled sheep anti-rabbit IgG could detect 1 ng of poly rI-rC. This assay could also detect the presence of double-stranded RNA in Semliki Forest virus-infected chick fibroblasts and can be used as an assay for interferon activity.

Detection of virus-specific immunoglobulins using a doubly labeled fluorescein-125I antibody.

Commercially prepared fluorescein-labeled antihuman antibodies were labed with 125I and used to compre specific herpes simplex virus antibody titers as determined by indirect fluorescent antibody and radioimmunoassay techniques. Total virus-specific immunoglobulin and virus-specific immunoglobulin G titers did not vary by more than one twofold dilution when compared by the two methods.

Comparison of solid-phase radioimmunoassays for baculoviruses.

The sensitivity and cross-reaction of four solid-phase radioimmunoassays (RIA) for Trichoplusia ni nuclear polyhedrosis virus containing singly enveloped virions were investigated. The detection limits of each assay were as follows: Indirect RIA, 5 ng of dissolved polyhedron antigen; direct RIA, 50 ng; indirect sandwich RIA, 200 ng; and direct sandwich RIA, 300 ng. The indirect and indirect sandwich RIAs showed considerable cross-reaction with other baculovirus antigens, but the direct and direct sandwich RIAs showed cross-reaction with only one closely related baculovirus. When microtiter plates used for the solid phase were pretreated with bovine serum albumin, nonspecific binding of labeled antibodies was reduced to a minimum. Antibodies prepared by an immunoadsorption procedure showed greater specific binding than antibodies prepared by ammonium sulfate precipitation of the immunoglobulin fraction. Highly contaminated antigen could not be detected by the indirect RIA, but the direct sandwich RIA was unaffected by antigen contamination. Antigen making up 0.0025% (wt/wt) of a sample of bird droppings could be detected by the direct sandwich RIA.

Comparison of the genomes of two sympatric iridescent viruses (types 9 and 16).

A map of the sites in the genome of Costelytra zealandica iridescent virus (CzIV), using the restriction enzymes BamHI, KpnI, and PstI, showed the genome size to be 170.2 kbp in length. It was found that the genome was cyclically permuted and that 39% of the genome of CzIV contained repetitive sequence elements. The genome was found to hybridize with the genome of another iridescent virus, type 9 (WIV), in DNA-DNA hybridization experiments. A region of the WIV DNA genome (23.4 kbp) did not hybridize with CzIV DNA and this region is similar in size to the total genomic size difference between CzIV and WIV (22.4 kbp). A unique repeat sequence from iridescent virus type 6 (CIV) was shown to be present in the genome of WIV but not that of CzIV. Finally, the positions of the major capsid protein genes, VP53 and VP52, in the restriction enzyme maps for type 16 and type 9 respectively were determined.

Purification and properties of a toxin isolated from Mortierella wolfii.

A substance toxic for mice has been extracted from the washed mycelia of Mortierella wolfii. This toxin could also be demonstrated in culture fluids after spontaneous autolysis of the fungal hyphae. Purification of the toxin was carried out using Sephadex chromatography (G-200, G-50) and resulted in a single band on sodium dodecyl sulfate polyacrylamide gels. Properties of the toxin included heat lability at 60 C, trypsin sensitivity, activity over a wide pH range, and a molecular weight of 23,600. The mean lethal dose for 16-week-old male mice was 3.3 mug of protein.