- Volume 38, Issue 1, 1978

VSV defective interfering particles of various sizes and from several independent sources frequently contain plus and minus strand RNA. In many cases some of the complementary strands are covalently linked as snap-back molecules. Infectious particles on the other hand package little or no plus strands. Snap-back molecules from the three different sources examined so far vary in size but appear to conform to the same overall linear duplex structure with cross-links at the ends only. They each contain a base sequence which is a subset of the next larger one and appear to correspond to unique sequences in the L cistron of the genome. Possible origins for these snap-back molecules are discussed.

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.

Interferon derived from the human lymphoblastoid cell line, Namalva, was fractionated by antibody affinity chromatography into two antigenically distinct interferon subspecies. At least 13% of the total Namalva interferon activity possessed the F antigenic determinant found on human interferon derived from fibroblast cultures, while the bulk of the Namalva interferon activity had the Le antigenic determinant characteristic for human leukocyte interferon. The separated Le and F subspecies of Namalva interferon differed in the degree of their heterospecific activities on bovine cells. The Le moiety resembled crude leukocyte interferon in that it was highly active in bovine cells. The F component of Namalva interferon showed a lower degree of activity in bovine cells, thus resembling crude fibroblast interferon. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by isoelectric focusing, crude Namalva interferon qualitatively resembled crude leukocyte interferon.

A study using three inbred lines, Reaseheath C, HPRS-synthetic E and RPRL-7-2, was made to investigate the inter-relationship between the genes that regulate antigenic expression of the subgroup E endogenous virus and those that control susceptibility to infection with exogenous viruses of subgroups A, B and E.

It was found that the tumour virus e (tve) and group specific (gs) antigen loci are linked, as indicated by the non-random association between the genes at the two loci. On the other hand, the genes at the other three loci, tva, tvb and gs, segregate and recombine at random in accordance with Mendel’s second law. That tvb and tve are two independently segregating loci, was also confirmed in this study. Furthermore, the evidence that the tve locus, while segregating independently of the tvb locus, is linked with the gs locus upheld the hypothesis of its existence contrary to the views of other workers. This study suggests that the gs locus could be used as a marker for the detection of the tve locus in chicken lines that lack the Ie gene. Also, it has been shown that the Ie gene is not identical to the gs + gene contrary to our earlier hypothesis.

Three polypeptides with mol. wt. 100 (P100), 80 (P80) and 65 (P65) × 103 were found in calicivirus infected cells. P100 and P80 were present in sub-molar amounts compared with P65 and no precursor product relationship between the three polypeptides could be demonstrated using pulse-chase experiments or selective inhibitors of protein synthesis and of proteases. In the presence of protease inhibitors a polypeptide with mol. wt. 120 × 103 (P120) was demonstrated which appeared to be the precursor of P100. Possible mechanisms of translation in the caliciviruses are discussed.

Nodamura virus, a small non-enveloped RNA virus, contains two species of RNA sedimenting at 22S (RNA-1) and 15S (RNA-2), a single major polypeptide of mol. wt. 40 × 103 and two minor polypeptides, of mol. wt. 38 and 43 × 103. Evidence is presented that the two RNA species are in the same particle. Although extraction of the virus with SDS-phenol yields the two species of RNA as separate entities, gentle treatment of the virus with guanidine and low concentrations of SDS releases the RNA as a 27S component which contains both RNA-1 and RNA-2 together with a trace of protein. It seems likely that the two RNA species replicate separately because double stranded molecules corresponding to the single stranded RNA-1 and RNA-2 molecules were present in BHK cells infected with the virus.

The results of analyses of fowl plague virus-specific RNA and protein synthesis in infected chick embryo fibroblasts incubated in amantadine hydrochloride are reported. They indicate that provided amantadine is present from the time of virus addition no expression of the virus genome occurs and that the synthesis of even the first detectable transcripts catalysed by the polymerase of the infecting virus particles is prevented. In agreement with previous reports it is concluded that amantadine prevents an unknown event which occurs immediately following virus infection.

This study investigated the synthesis of membrane antigen (MA) as well as virus capsid antigen (VCA) and early antigen (EA) in Daudi cells which had been superinfected with the P3HR-1 strain of Epstein-Barr virus (EBV) and then treated with trypsin to remove initially absorbed MA-positive material from the cell surface. Synthesis of MA, VCA and EA was completely inhibited by puromycin. A marked reduction in the frequency of MA positive cells was observed in superinfected cells cultured in the presence of either cytosine arabinoside (Ara-C) or phosphonoacetate (PA); however, a small fraction of MA synthesis occurred, suggesting an inhibitor insensitive component in MA. A differential absorption of EBV antibody-positive human serum with the Ara-C treated or untreated infected cells detected two antigenically different components in MA: early (Ara-C insensitive) and late (Ara-C sensitive) MA.

Ultraviolet (u.v.) light-irradiation of human cytomegalovirus (HCMV) resulted in differential inactivation of virus capacities, e.g. induction of cell rounding, early antigens (EA), nuclear inclusion, HCMV DNA synthesis, cellular DNA synthesis, HCMV-specific DNA polymerase, cellular DNA polymerases and plaque production, while the capacity of HCMV to penetrate cell nuclei was not critically impaired. These results indicated that the virus-coded functions expressed after infection were responsible for all these events except for HCMV-induced stimulation of cellular RNA synthesis which was enhanced by irradiation of the virus at a low dose of u.v. light (6600 ergs/mm2). In these experiments phosphonoacetic acid was effectively utilized to detect EA formation by immunofluorescent staining and to differentiate cellular DNA synthesis from virus DNA synthesis.

The DNAs of rabbitpox, vaccinia, cowpox, ectromelia and fowl pox virus, all grown in the same host system, were cleaved with restriction endonucleases HindIII, BamI and EcoRI. The resulting digestion products were separated by electrophoresis on agarose slab gels. From the similarities and dissimilarities of the migration profiles obtained, it was concluded that vaccinia and rabbitpox virus are genetically the most closely related viruses investigated, although their DNA cleavage patterns are clearly distinguishable. Cowpox and ectromelia virus both show about the same degree of relatedness to each other as to rabbitpox and vaccinia virus. Fowl pox virus, which belongs to the genus Avipoxvirus, contains the largest genome examined (approx. 160 × 106 daltons) and the cleavage patterns of its DNA show no similarities with those of orthopoxvirus DNAs, thus indicating a very low degree of genetic relatedness. We believe that restriction analysis is a useful method for the identification and classification of poxvirus isolates.

Spleen cultures from various strains of mice were infected in vitro with murine cytomegalovirus (MCMV). Infectious centres were established in a small proportion (not greater than 1%) of the cells. Virus could be rescued from these cells by co-cultivation with syngeneic or allogeneic fibroblasts, but the frequency of rescue could not be altered by incubation with cyclic nucleotide analogues, iododeoxy-uridine, cortisol, or allogeneic spleen cells. In addition a smaller fraction of the cell population, possibly a sub-population of the infectious centres, replicated virus spontaneously. The presence of mitogens did not affect these interactions qualitatively or quantitatively. A third response to infection was an inhibition in DNA synthesis, which was suffered by unstimulated cultures and by cells transformed by concanavalin A and bacterial lipopolysaccharides, although overall cell viability was maintained. This response was also mediated by u.v.-inactivated virus.

A combinatorial analysis of the products of DNA cleavage by restriction endonucleases permits an estimation of the sequence homology between closely related linear DNAs. The main assumption to be made is that fragments of identical length represent equivalent sections of the genomes. All patterns of common and varying restriction sites compatible with the results of the electrophoretic analysis of fragment sizes can be enumerated, each yielding an estimate of the proportion of sequences of restriction site length common to both DNAs. From this, sequence homology is derived by using a model of randomly distributed point mutations relating the two genomes. Possible limitations of the approach are discussed.

A previous report in this Journal of a computer method for assessing the size of a protein from its amino acid composition, and its application to virus protein data, contained an error in the published arithmetical formula. Persons using the incorrect formula would not have obtained incorrect estimates of possible protein sizes, but would have obtained a more equivocal set of possible sizes.

The isoelectric point (pI) of the p30 polypeptide of members of the three known classes of mouse C-type endogenous viruses was determined both by column and by thin-layer gel isoelectric focusing. Each class was found to be characterized by a particular variant of p30 (isop30), with pI values of 6.1 for class I (ecotropic), 5.7 for class II (xenotropic), and 5.5 for class III (NZB, NIH, ATS124, also xenotropic). The 6.1-isop30 was found as a minor component of rat-grown NZB virus and of a number of laboratory strains of mouse C-type viruses.

A plaque assay for iridescent virus type 22 (from Simulium sp.) using Spodoptera frugiperda cells has been devised, and the kinetics of growth of the virus in this cell line have been determined. The virus particle/p.f.u. ratio was 75 ± 8, and the p.f.u./TCID50 ratio was 0.56 ± 0.11.

This report offers a description of a quantitative micro-complement fixation method (Cikes, 1975) for detecting human wart virus antigens and their specific antibodies, and proof of the specificity of the reactions being detected. The increased sensitivity demonstrated by chromium-release measurement is compared to the results of visual interpretation of complement fixation.

The replication of virulent African swine fever virus (ASFV) in cultures of monocytes and macrophages derived from pig bone marrow (PBM) and pig leukocyte (PL) cells was investigated by light microscopy, immunofluorescence, haemadsorption and infective virus release. Monocytes showed a high rate of infection and complete destruction within 2 to 3 days, whereas macrophages had only a very low level of infection and survived to form persistently infected cultures. These observations may explain the decrease in sensitivity of PBM and PL cells for ASFV assay after extended periods of incubation and suggest that the macrophage may be one of the cell types concerned with virus persistence in the pig.

The major influenza A virus structural antigens, matrix protein, nucleoprotein, haemagglutinin and neuraminidase were measured rapidly and accurately using a rocket immunoelectrophoresis technique. Virus was disrupted with 1% (w/v) sodium sarcosyl and electrophoresed into agarose containing specific antiserum to the individual virus structural proteins in 0.05 m-barbitone buffer, pH 8.6, for 1 to 4 h. For haemagglutinin antigen assays statistical analysis indicated that the coefficient of variation within an immunoelectrophoresis plate was 8.0% for antigen concentrations in the range 15 to 40 µg/ml protein. For haemagglutinin and matrix protein the method was sufficiently sensitive to measure concentrations of antigens as low as 1.5 and 2.0 µg/ml respectively. By incorporation in the agarose of mixtures of antisera against specific antigens of the virus, haemagglutinin, matrix or nucleoprotein could be assayed simultaneously.