Physical and genetic map of the Wiseana nucleopolyhedrovirus genome.

Wiseana nucleopolyhedrovirus (NPV) is the major pathogen of the New Zealand endemic pasture pest, Wiseana spp. To characterize this potential biological control agent, the genome of a virus isolated from Wiseana signata was purified and cloned. The complete genome was cloned as BamHI or HindIII restriction fragments, which were mapped by Southern hybridization and restriction analysis. To verify the physical map, the junctions between all HindIII fragments were confirmed by sequencing. The viral genome was estimated to be 128 kbp. Sequence data generated at the termini of cloned restriction fragments were compared to sequence databases to identify putative gene homologues. Seventeen putative ORFs, which were homologous to other baculoviral sequences, were identified. These putative ORFs were located on the Wiseana NPV physical map and their distribution was compared to genetic maps of NPVs isolated from Autographa californica, Orgyia pseudotsugata and Lymantria dispar. Although the virus from W. signata was significantly different from these other NPVs, a core region of the viral genome was conserved.

A novel capsid expression strategy for Thosea asigna virus (Tetraviridae).

This paper presents evidence that Thosea asigna virus (TaV) has a unique capsid expression strategy and is a member of the Nudaurelia beta-like genus of the Tetraviridae. Electron microscopy of TaV particles indicated a 38 nm, T = 4 icosahedral capsid similar in structure to that of Nudaurelia beta virus (NbetaV). TaV particles have a buoyant density of 1.296 g/cm3 in CsCl and consist of two capsid proteins of 56 and 6 kDa. The virus genome contains a genomic RNA molecule of 6.5 kb and a subgenomic molecule of 2.5 kb. Northern blotting of TaV RNA indicated a genomic organization similar to that of NbetaV. The capsid gene of TaV is carried on both the genomic and subgenomic RNA molecules, while the RNA polymerase gene is present only on the genomic RNA. Cloning and sequencing of the TaV capsid gene identified an open reading frame that could potentially encode a capsid precursor protein of up to 82.5 kDa. The N-terminal sequences of the capsid proteins were compared with the nucleotide sequence of the capsid open reading frame. The sequences indicate that the pre-protein is cleaved at two positions to produce the 56 and 6 kDa capsid proteins as well as a predicted third protein that was not detected in the mature virion. Phylogenetic analysis of the capsid proteins indicated that TaV is more closely related to NbetaV than to the Nudaurelia omega-like viruses. The eight beta-sheets that make up a jelly roll structure in the TaV capsid protein were identified by computer analysis.

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.

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.

Examination of New Zealand's endemic Wiseana nucleopolyhedrovirus by analysis of the viral polyhedrin gene.

Insects of the genus Wiseana (Lepidoptera: Hepialidae) are major agricultural pests in New Zealand. Singly enveloped nucleopolyhedroviruses (SNPVs) isolated from three of the seven described Wiseana species have potential as biological control agents. As part of an effort to characterise the Wiseana SNPV genome the polyhedrin gene was cloned and the nucleotide sequence determined. The gene sequence was used, in conjunction with morphological and restriction endonuclease analysis, to compare isolates from different sites and species of Wiseana. Heterogeneity was detected within a single site, as well as between SNPV from separate Wiseana species. The extent of divergence between the nucleotide sequences was small enough, however, to consider three SNPVs from W. signata, W. cervinata and W. umbraculata as different strains of a single SNPV species. This improves the likely practicability of developing a single viral agent to control this pest complex. In addition, the virus polyhedrin gene sequence was used to estimate the phylogenetic relatedness of a W. signata SNPV to 16 other NPV from diverse insect genera. These comparisons suggest the Wiseana SNPV was unique within the Baculoviridae, but was more closely related to the group II NPVs.

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.

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.

A comparison of the structural polypeptides of three iridescent viruses (types 6, 9, and 16) and the mapping of the DNA region coding for their major capsid polypeptides.

The iridoviruses from Wiseana cervinata (WIV, type 9), Costelytra zealandica (CzIV, type 16) and Chilo suppressalis (CIV, type 6) were compared by SDS-PAGE and Western protein blotting for antigenic determinants. The major capsid proteins were isolated and oligonucleotide probes were synthesized from the partial amino acid sequences. The DNA regions coding for the major capsid proteins of WIV (VP52), CzIV (VP53) and CIV (VP50) were located by hybridization of the oligonucleotide probes to blots of the viral DNA. The major capsid protein was used as the zero point for the proposed linearized maps of these viruses. Using antibody and 125I-labelling, several proteins were identified as being on the surface of the virion. It was also shown that CIV was not as antigenically distinct from these two viruses as previously reported.

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.

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.

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.

Changes in anti-DNA antibody affinity during exacerbations of systemic lupus erythematosus.

The level and average functional affinity of anti-DNA antibody were measured retrospectively in successive serum samples from 5 patients with systemic lupus erythematosus. In 2 patients, severe flares of disease activity were associated with an increase in the level and average functional affinity of anti-DNA antibodies. Three patients who did not experience significant flares in disease activity exhibited more constant levels of antibody of lower average functional affinity. The appearance of antibody of high functional affinity during disease exacerbations suggests that such antibodies may be pathogenic.

Rotavirus infection in a small community.

Serial titres of rotavirus specific IgG and IgM have been measured in children and adults living in a small community over a 2 1/4-year period. In all age groups the mean titres of rotavirus specific IgG and IgM rose and fell in parallel with the changes in frequency of gastroenteritis symptoms in the community but after the time when respiratory symptoms reached their peak. Gastroenteritis symptoms were seen most commonly in the children but were also frequent in adults, especially the women. Titres of rotavirus specific IgG changed with age, increasing through childhood into early adult life, but decreased thereafter only to increase again in those over the age of 50 years. Females had higher levels of IgG in all age groups but especially among the children and 30-49-year-old women. The high levels of IgG did not protect the young adults from symptomatic gastroenteritis. Detectable levels of rotavirus specific IgM occurred in all age groups but more commonly in children aged under 10 years and in young adults. Raised levels of IgM were uncommon in the elderly, who rarely suffered gastroenteritis symptoms. An epidemiological model is proposed in which the older members of the community act as a reservoir of rotavirus, passing the infection to the children, who then infect the young adults.

Isolation of rotavirus from calves, foals, dogs and cats in New Zealand.

The incidence of rotaviruses in calves, foals, dogs and cats in the Dunedin urban and rural areas was investigated using electron microscopy and enzyme-linked immunosorbent assays. Of the 283 faecal specimens examined, 26% were positive for rotavirus. Comparison of the genetic electropherotypes was made by separating the viral dsRNA segments using polyacrylamide gel electrophoresis. It is possible that rotavirus infection is a zoonotic disease.

Sequence relationships between the genome segments of human and animal rotavirus strains.

The sequence relationships of a range of cultivable and noncultivable human and animal rotaviruses were investigated by hybridization of rotavirus cDNA probes to genomic RNAs immobilized on diazobenzyloxymethyl paper. Under conditions of low stringency (34% base mismatch tolerated) most genome segments exhibited partial homology except for genes 4 and 5. In contrast, under more stringent conditions of hybridization in which no more than 8% base mismatch was tolerated, few segments exhibited homology. Generally the human and animal rotaviruses were found to possess distinct nucleic acid sequences that exhibit only a low order of sequence relatedness. These results are consistent with the notion that both cumulative changes in nucleic acid sequences and the interchange of segments may be involved in the evolution of distinct rotavirus strains.

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.

Antibodies against double-stranded RNA in patients with rheumatoid arthritis, osteoarthrosis and Paget's disease of bone.

Using a liquid phase radioimmunoassay to detect antibodies to 3H-labelled double-stranded RNA the premise that rheumatoid arthritis and Paget's disease of the bone may be associated with a chronic virus disease was examined. About 33% of patients with rheumatoid arthritis had antibody levels above the normal range and 11% had antibody levels below the normal range of controls (blood bank donors). The low binding activities were attributed to the action of a nuclease that degraded the dsRNA. Some patients with Paget's disease of bone had higher binding activities than the normal range and similar binding activity was also found in patients with osteoarthrosis. The increase in antibodies to double-stranded RNA did not correlate with increasing age.

Parainfluenzavirus upper respiratory tract illnesses in partially immune adult human subjects: a study at an Antarctic station.

Outbreaks of respiratory tract illnesses (RTI) in adult humans during October and November 1975 at McMurdo Station, Antarctica, were investigated by viral isolation and serologic procedures. The recovery of viral agents was enhanced by use of cell cultures in the field. Recoveries of parainfluenzaviruses types 1 and 3 and rhinoviruses were made from 10 of 39 nasal washings. Parainfluenzaviruses types 1 and 3 accounted for 50 and 30 per cent, respectively, of the total viruses recovered during October and November 1975. Acute and convalescent sera collected from 32 adult humans were examined for antiviral antibody by hemagglutination inhibition (HI) and radioimmunoassay (RIA) techniques. Serologic responses (HI and RIA) confirmed that parainfluenzaviruses were the predominent cause of RTI at McMurdo Station during that time. The temporal relationship between parainfluenzaviral diseases occurring in US communities and at McMurdo suggests that these viruses are transported to the Antarctic by personnel originating within the US. Standardization of the RIA allowed sequential assay of large numbers of sera using multiple preparations of radiolabeled indicator antibody, while ensuring the reproducibility of antiviral antibody titers to within one twofold dilution between subsequent labeled antibody preparations. The RIA detected both lower levels of virus specific antibody and more serologic responses than could be detected by HI.

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.