Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure.

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.

Pernin: a novel, self-aggregating haemolymph protein from the New Zealand green-lipped mussel, Perna canaliculus (Bivalvia: Mytilidae).

A protein, designated pernin, found in the New Zealand green-lipped mussel, comprises almost all of the protein in cell-free haemolymph. It occurs as large, aggregate structures of several hundred units resembling small virus-like particles. Pernin is a non-pigmented, glycosylated protein, composed of 497 amino acids, which has an estimated molecular mass of 60 kDa. It is exceptionally rich in histidine (13.7%) and aspartic acid (12.3%), amino acids both known to participate in the binding of divalent metal cations. In addition, pernin has serine protease inhibitor activity, likely due to a sequence of eight N-terminal amino acid residues, separated from the remainder of the protein via a histidine-aspartate spacer. The pernin monomer comprises three regions of obvious sequence duplication. These make up approximately 95% of the pernin molecule and have sequences clearly homologous to the active-site domain of Cu-Zn SODs (superoxide dismutases). Despite several of the metal ion co-ordinating histidine residues being retained, pernin contains no Cu or Zn. It is, however, associated with Fe with an apparent stoichiometry of 1 atom of Fe to 6 molecules of pernin. Since pernin has no demonstrable SOD activity, these SOD-derived sequences presumably have been modified for another function.

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.

The production of cricket paralysis virus in suspension cultures of insect cell lines.

The yields of cricket paralysis virus from two insect cell lines propagated in suspension culture were examined. Although Trichoplusia ni (TN368) cells produced more virus per cell than Drosophila line 2 (DL2) cells, the smaller DL2 cells reached a higher cell density. Thus the difference in production between TN368 and DL2 cultures was minimal. In mixed infections of Flock House virus and cricket paralysis virus (CrPV), CrPV was the predominant virus produced despite being present at a much lower multiplicity of infection.

Manawatu virus: a nodavirus isolated from Costelytra zealandica (white) (Coleoptera: Scarabaeidae).

An insect virus, called Manawatu virus (MwV), was isolated from a larva of the New Zealand grass grub Costelytra zealandica (White) (Coleoptera: Scarabaeidae). MwV was serologically related, but not identical, to several insect nodaviruses. The single capsid protein of MwV was 40,000 MW, the same as black beetle virus (BBV), but virus particles had a different electrophoretic mobility from BBV. The bipartite RNA genome, like other nodaviruses, consisted of two species of MW 1.1 and 0.46 million. MwV particles sedimented at 142 S and had an estimated density in neutral CsCl of 1.366 g/ml compared with 1.352 g/ml for BBV. The serological and physico-chemical properties, compared with other nodaviruses, indicate that MwV is unique.

The estimation of virus density in isopycnic cesium chloride gradients.

Methods for estimating the density of virus particles after isopycnic centrifugation in cesium chloride gradients were examined. If solutions were prepared in different buffers, the standard tables relating refractive index and density, based on solutions prepared in water, did not give consistent estimates even if corrections were made for the greater refractive indices of the buffers. Depending on the method and buffer used, estimates of the density of cricket paralysis virus, an insect picornavirus, differed by as much as 0.05 g/ml. However, consistent estimates were obtained if separate regression equations were derived to relate refractive index and density for each different buffer solution of cesium chloride. This has wider implications and could account for density differences or variations in estimates reported for many other viruses.

Flock House virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae).

The physico-chemical properties of Flock House virus (FHV) were examined. FHV was shown to have a single-stranded RNA genome divided between two species of MW 1.1 and 0.46 million; the genome was infectious. FHV has a single, coat protein of 43,000 MW. The virus sediments at 142S and has a density of 1.351 g/ml in CsCl at pH 7. These properties indicate that FHV is a nodavirus but it is distinguishable from Nodamura virus and black beetle virus by differences in electrophoretic mobility, the size of the coat protein, particle density and stability in CsCl.

Factors affecting the use of immunoosmophoresis for the detection of two insect riboviruses.

Some of the factors affecting the formation of precipitin lines during immunoosmophoresis were investigated using two unrelated insect riboviruses, Flock House virus (FHV) and cricket paralysis virus (CrPV). Precipitin lines formed in under 90 min. Concentrations of 200-400 ng/ml of FHV were routinely detected but occasionally the end-point was as low as 50 ng/ml. With CrPV, 1500 ng/ml was the routine detectable level. The sensitivity of the assay was largely unaffected by the presence of insect homogenate. Maximum detection of virus was obtained when the antigens and antibodies were at optimal proportion and excess of either reagent resulted in the blurring of precipitin lines. Immunoosmophoresis proved to be a rapid and inexpensive method of detecting negatively charged viruses in crude insect extracts and can, in some cases, have a sensitivity approaching that of more sophisticated serological techniques.

Naturally occurring IgM antibodies to a small RNA insect virus in some mammalian sera in New Zealand.

Antibodies to cricket paralysis virus were demonstrated in sera from a pig, a horse, and numerous cattle in New Zealand. The reactions in immunodiffusion tests were variable, but significant and consistent reactions were obtained with these sere in virus neutralization assays using Drosophila cells. The sedimentation coefficient of the antibodies was 19S, and their activity was destroyed by treatment with 0.1 M 2-mercaptoethanol, indicating that they were of the IgM class.

End-point dilution and plaque assay methods for titration of cricket paralysis virus in cultured Drosophila cells.

Cricket paralysis virus, an insect picorna-like virus, caused a distinct c.p.e. in cultured Drosophila melanogaster cells, allowing the development of titration methods based on end-point dilution or plaque assay methods. The end-point dilution (TCD50) method is more sensitive and economical than plaque assays and is easily scored. The data indicate a minimum infectivity/particle ratio of about 1/2000.

Particles produced during a mixed infection by two tobamoviruses contain coat proteins of both viruses.

Particles from plants mixedly infected with two tobamoviruses, whose particles differ in density, were all of a single, intermediate density, suggesting that they contain both coat proteins and that the proteins occur in the same proportion in all particles. Plants inoculated with the genome-containing particles of one of the tobamoviruses together with the noninfective, short, coat protein messenger-containing particles of the other yielded only particles with the density of the first;.there was no evidence of complementation.

Serological relations between twelve small RNA viruses of insects.

Serological tests were done to examine the relationships between twelve picorna-like viruses of insects. The results of the tests indicated that the majority of the viruses are unrelated. However, cricket paralysis virus, isolated from Australian wild field crickets, appeared to be identical to Drosophila C virus, independently isolated in France. Cricket paralysis virus was infective for adults of Drosophila melanogaster and its infectivity towards Galleria melonella was neutralised by Drosophila C virus antiserum. It is therefore concluded that cricket paralysis virus and Drosophila C virus are very closely related if not identical.

On the nature of the difference in the densities of the particles of two tobamoviruses.

Particles of sunnhemp mosaic virus (SHMV) are denser than those of the U2 strain of tobacco mosaic virus (T2MV) when their densities are estimated by equilibrium centrifugation in gradients of either cesium chloride or Metrizamide; in cesium chloride the densities are 1.318 and 1.307 g/ml, and in Metrizamide they are 1.249 and 1.240 g/ml. Experiments with particles reassembled from homologous or heterologous mixtures of the RNAs and coat proteins of the viruses show that the difference in their densities is determined by their coat proteins. The disassembled coat proteins of the two viruses have the same density, but polymerized SHMV protein is less dense than polymerized T2MV protein. Particles reassembled from homologous or heterologous mixtures of the RNAs and coat proteins of the viruses have the density of the nucleoprotein particles used as the source of protein. The density difference of the two virus nucleoproteins therefore reflects the different behavior of the two proteins on assembly with RNA.

Cricket paralysis virus replicates in cultured Drosophila cells.

Drosophila cells infected with cricket paralysis virus yielded normal virus particles as shown by their infectivity and by physical and serological tests; the paricles had the same sedimentation coefficient and density as the virus particles used as inoculum, and were precipitated specifically by an antiserum to those particles.

Events occurring during the replication of bacteriophage T4 DNA.

Ten temperature-sensitive mutants of T4D have been examined to find the times in the lytic cycle at which a shift to a restrictive temperature no longer exerts a lethal effect. Nine of these mutants play a role in phage DNA synthesis. The results of these experiments suggest that the products of three of the genes tested-genes 42, 44, and 45-have control or complexing functions in addition to any enzymatic ones. The data also indicate that the products of genes 30 and 41 may act cooperatively. The properties of mutants of gene 43 (DNA polymerase) and gene 56 (dCTP-dUTPase) suggest that the production of a certain length of concatameric DNA is necessary before viable phage can be produced.