- Volume 45, Issue 3, 1979

The structural polypeptides of accepted species and recently isolated members of the genus Orthopoxvirus have been examined by SDS-polyacrylamide gel electrophoresis. The viruses shared many polypeptides but some differences were found. The viruses could be divided into a vaccinia group (including buffalopox, ‘Lenny’ and MK-10), an ectromelia group (including elephant virus and Moscow virus), cowpox, camelpox and monkeypox. Minor differences were found in the polypeptides of monkeypox virus strains from human and monkey outbreaks. Controlled degradation of virions showed that the polypeptides which enabled the viruses to be differentiated were located in the surface and sub-surface layers. The cores of the viruses all gave the same complex polypeptide pattern.

Fluctuations in the expression of measles virus surface-associated antigens in persistently infected human Lu 106 cells were analysed by the use of human sera which preferentially reacted with the haemolysin or haemagglutinin component. The variations observed correlated with the proportion of cells expressing surface-antigen, as examined in population analysis by indirect immunofluorescence, a radio-labelled antiglobulin technique and a cytotoxicity assay. Microfluorometric analysis revealed no changes in antigen expression at the single-cell level. The number of cells that were positive by immunofluorescence among exponentially growing, low density cells remained relatively constant. These cells were more susceptible to cytotoxicity mediated by both antisera and complement than cells seeded at high density and kept in the stationary phase. The percentage of fluorescent cells among the latter cells gradually decreased. Thus cytotoxic susceptibility was related to the proportion of the total cell population that was antigen-bearing, rather than to variations in the expression of antigen at the single-cell level. In mitotic cells, polarization of antigen, as measured by indirect immunofluorescence of pre-fixed cells, was frequently seen. Often only one of the daughter cells expressed surface antigen. The results imply that cells in stationary phase may lose antigen from their surface, possibly by shedding, and furthermore that re-expression would demand a new cell cycle.

Chicken fibroblasts and MDCK cells were infected with influenza virus labelled with either 3H-uridine or 14C-amino acids, and the location in infected cells and properties of input virus-labelled structures were studied. Input virus RNA and protein were found in the cytoplasm of nuclei 1 h p.i. A part of the intranuclear parental structures was associated with chromatin while the other part could be extracted from nucleoplasm by 0.16 m-NaCl and represented free ribonucleoprotein (RNP) particles. These RNPs sedimented in glycerol velocity gradients at 40 to 70S, very similar to cytoplasmic RNPs, but differed distinctly from them in buoyant density. The bulk of cytoplasmic RNPs after fixation with formaldehyde banded in CsCl at 1.34 g/ml while nucleoplasmic RNPs banded at 1.39 or 1.41 g/ml. RNPs isolated from virions and infected cells contained the NP polypeptide which was revealed by SDS-PAGE analysis as a double band. The ratio of the two bands varied in cytoplasmic and nucleoplasmic RNPs, the lower band being dominant in cytoplasmic but not in nucleoplasmic RNPs. In addition, cytoplasmic RNPs were phosphorylated. The possible significance of intracellular RNP modifications for virus replication is discussed.

Various degrees of permissiveness for NDV have been described in different cell lines. In the present work three systems were investigated: bovine kidney (MDBK) cells, chick embryo (CE) cells and mouse L cells producing 50 to 100 p.f.u., 2 to 10 p.f.u. and less than 0.1 p.f.u./cell, respectively. Analysis of the radioactive virus messenger RNAs (vmRNAs) accumulating in actinomycin D-treated cells throughout the infection cycle revealed that, except for the absence of one 18S vmRNA species in L cells, all vmRNA components were formed in the three cell types, although variations occurred in their total and relative amounts from one cell type to another. Kinetic studies of vmRNA synthesis confirmed the absence of one 18S vmRNA species in L cells and also showed that the labelling rate of this vmRNA component is higher in CE cells. Virus proteins synthesized in the infected cells were labelled with 14C-amino acids and analysed by polyacrylamide gel electrophoresis. All the major NDV polypeptides were formed as expected in both CE and MDBK cells but only traces were detected in L cells. In contrast in a cell-free translation system from wheat germ, all of the DNV major proteins were synthesized using RNA extracted from the three infected cell types. Moreover, the total radioactivity incorporated into NDV proteins was twice as great with CE cell RNA and five times as great with L cell RNA than with equivalent quantities of RNA from MDBK cells.

A comparison has been made of some of the biochemical and serological characteristics of five isolates of foot-and-mouth disease virus (FMDV), serotype A. Three of the viruses have been assigned to the same subtype, A22; the other two belong to different subtypes, A5 and A24. RNA competition hybridization and two-dimensional electrophoresis of the oligonucleotides produced by ribonuclease T1 showed that the three A22 viruses formed a group which could be distinguished from the A5 and A24 viruses. However, the three A22 viruses showed some differences by both tests. Analysis of the virus polypeptides by polyacrylamide gel electrophoresis methods also distinguished the A22 viruses as a group distinct from the A5 and A24 viruses, but small differences within the A22 group were observed using electrofocusing techniques. Serological differences were observed between the viruses using complement fixation tests and by competition radioimmunoassay with antisera obtained from guinea pigs infected with these viruses. The greatest similarity occurred between the viruses previously subtyped as A22, with A5 and A24 being distinct from the A22 group and from each other. The relationship of the biochemical and serological data is discussed.

Mount Elgon bat virus given intracerebrally readily killed mice up to 12 days of age (1 to 4 p.f.u./LD50). Virus in doses of 10 to 106 p.f.u. killed 40 to 80% of weanling mice of both sexes and up to 20% of adult females and 40 to 80% of adult males. Clearance of brain infectivity in the resistant mice coincided with the appearance of circulating virus neutralizing antibody which occurred earliest in the adult female mice. Immunosuppression of adult mice with cyclophosphamide resulted in the death of all the virus-inoculated mice of both sexes. Virus in these mice reached tenfold higher titres and their brains contained 30- to 60-fold more interferon than the brains of infected mice not given cyclophosphamide. Interferon was apparently without effect on the outcome of infection in the brains of resistant mice and its synthesis reflected the extent of virus growth.

In culture cells productively infected by adenovirus a high mol. wt. form of DNA is synthesized which is known to represent, at least in part, virus DNA integrated into cellular DNA. We found that the synthesis of this high mol. wt. DNA and the other DNA size classes can strongly and differentially be influenced by altering the metabolic state of the cells. The effects of different rates of cell growth were tested in this respect as well as arginine deprivation as opposed to application of complete growth medium. Synthesis of virus high mol. wt. DNA and unit genome length DNA is enhanced in actively growing as compared to resting Ad5-infected HeLa cells. Under arginine deficiency, in resting Ad5-infected HeLa cells, integration of virus DNA sequences into cellular DNA is almost totally suppressed whereas virus unit genome length DNA is still synthesized. This differential effect is interpreted by the hypothesis that the formation of virus high mol. wt. DNA is a synthetic process that is independent of the unit size virus DNA replication, but coupled to the synthesis of a special form of cellular DNA that is less effectively shut off by the infection than cellular DNA in general.

Structural and virus-induced infected cell polypeptides of several strains of influenza B virus were examined by high resolution polyacrylamide gel electrophoresis and shown to be directly analogous to those of the influenza A viruses. Eight structural polypeptides, P1, P2, P3, HA1, HA2, NA, NP and M were observed in purified virus and at least two additional polypeptides, HA and NS could be detected in infected MDCK cells. The three P proteins plus NP were shown to be associated with RNA-dependent RNA polymerase activity and HA, HA1, HA2 and NA were shown to be glycosylated. Like the influenza A viruses, migrational differences of some of the infected cell polypeptides could be observed between different B strains. Investigation of a time course of virus replication failed to show any temporal control of protein synthesis in the infected cell.

Two mutually exclusive pathways of cytological change in the chloroplasts of Chinese cabbage cells infected with turnip yellow mosaic virus have been defined by light microscopic examination of individual infected protoplasts. In the first, all the chloroplasts in a cell became rounded and clumped together. This was followed by the development of a large vacuole, giving the chloroplast a sickled appearance. In the second pathway the chloroplasts became angular in outline before clumping, and subsequently fragmented to yield small pieces of chloroplast. Both these responses are controlled by the virus genome because some virus strains gave rise to the sickling response, while others cause fragmentation. Photosynthetic activity is a prerequisite for both pathways since they occur only when the protoplasts are illuminated, and both are inhibited by an inhibitor of photosynthetic electron transport.

We have successfully isolated and spread individual chromosomes of CHO-KI cells for electron microscopic karyotyping. Controlled preparation permitted a quantitative evaluation of the association between endogenous intracytoplasmic type A virus precursor particles and the centromeric region (kinetochores) of isolated chromosomes at prophase and metaphase. Our results suggest the transfer of type A particles from the cytoplasmic to the centromeric regions during early metaphase in conjunction with microtubule assembly at a time when the kinetochores are structurally mature and capable of binding microtubules. Preliminary comparable studies of the endogenous M432 virus propagated in murine cells support these findings. Our results are discussed with respect to mechanisms of intracellular movement of virus precursor particles and the interference with components of both the cytoskeleton and the mitotic apparatus.

Purified Drosophila X virus (DXV) particles have been analysed. They band at a density of 1.345 g/ml in CsCl. The virion proteins have been resolved into six major polypeptide species (mol. wt. 100000, 50000, 49000, 44000, 33000 and 27000) by polyacrylamide gel electrophoresis. The RNA sediments at 5S and 14S in sucrose gradients. The 5S RNA is sensitive to pancreatic RNase and the 14S RNA is resistant in its native form and sensitive after denaturation. The 14S RNA can be resolved into two equimolar fractions by polyacrylamide gel electrophoresis. The estimates of the mol. wt. of the two RNA species depends upon their structure. If they exist as double-stranded molecules their electrophoretic mobility compared to that of reovirus type 3 RNAs indicates for each species an average mol. wt. of 2.2 × 106.

Oat sterile dwarf virus (OSDV) subviral particles (SVPs) were purified from roots and stem bases of Lolium multiflorum. Electron microscopy of the preparations showed B-spiked SVPs typical of Fijiviruses (Fiji disease and maize rough dwarf-like viruses). Two sera with titres in gel-diffusion tests of 1/512 and 1/2048 were prepared against the SVPs. The sera did not react with the SVPs of maize rough dwarf and pangola stunt viruses when tested by gel-diffusion and immuno-electron microscopy. The first serum did not react with virus dsRNA or poly (1).poly(C) and the second serum had a titre of 1/8 with each of these. Poly-acrylamide gel electrophoresis of the nucleic acid from purified OSDV SVPs revealed ten dsRNA segments though only nine of these were resolved as separate bands in dsRNA extracted directly from infected plants. The genome pattern was broadly similar to but distinct from those of the known Fijiviruses. The RNA of the serologically related Arrhenatherum blue dwarf virus (ABDV), extracted directly from plants, appeared identical to that extracted directly from OSDV-infected plants. Both OSDV and ABDV were transmitted by the planthopper vector Javesella pellucida despite, in the case of OSDV, having been maintained in vegetatively propagated plants for 5 years.

Pangola stunt virus (PSV) subviral particles (SVPs) were purified from roots and stem bases of Digitaria decumbens (pangola grass); electron microscopy of the preparations showed B-spiked SVPs typical of Fijiviruses. An antiserum of geldiffusion titre 1/1024 was obtained. Serological tests confirmed that both the B spikes and the inner capsid of PSV SVPs were related to those of maize rough dwarf virus (MRDV), though PSV SVPs also carried specific determinants. Isopycnic centrifugation of PSV RNA showed that it is double-stranded, with a G + C content of 41 to 43%. Polyacrylamide gel electrophoresis showed that the RNA consisted of ten segments similar to those of MRDV, though segment eight was present in less than equimolar amounts.

Poliovirus particle RNA has been considered to have little secondary structure. Specific binding of poliovirion RNA by antibodies against double-stranded RNA (dsRNA) has been confirmed and further characterized by a radioimmunoassay using Staphylococcus aureus protein A to precipitate the nucleic acid-antibody complex. Competitive binding studies between virion single-stranded RNA (ssRNA) and poly(I).poly(C) demonstrated that the dsRNA effectively inhibited binding of radiolabelled poliovirion RNA by the anti-dsRNA antibodies but the virion RNA was a poor competitor of radiolabelled dsRNA. This indicates that both RNAs reacted with the same species of antibodies in the sera, but avidity of the antibodies for dsRNA was greater than for the poliovirion RNA.

Enveloped and naked frog virus 3 particles have been separated on sucrose gradients and their morphology checked by electron microscopy. Naked nucleocapsids sedimented at a density of 1.25 g/ml and enveloped virions at a density of 1.20 g/ml. The enveloped virions were found to possess an additional 58 K mol. wt. polypeptide. The ratio between the number of infectious particles, determined by plaque titration, and that of physical particles established by radioactive labelling as well as by electron microscopic counts, showed that the infectivity of the enveloped particles could be up to 150-fold greater than that of naked ones.

Orthopoxvirus DNA from representative strains of rabbitpox, vaccinia, monkeypox, variola, cowpox and ectromelia viruses was analysed by cleavage with restriction endonucleases HindIII, Xho1 or Sma1. Genome mol. wt. vary from about 120 × 106 for rabbitpox to about 145 × 106 for cowpox. Physical maps of cleavage sites are similar and characteristic for strains of the same Orthopoxvirus type. The distribution of HindIII sites suggests that an internal region of mol. wt. about 30 × 106 is highly conserved between Orthopoxvirus genomes although some type-specific differences occur within this region, especially with strains of ectromelia virus. Conservation of internal sequences is less marked following analysis with Xho1 although cleavages within this central region of particular genomes appear to represent a subset of preferred sites. Endonuclease Sma1 cleaves exceptionally infrequently and distinguishes variola, monkeypox, vaccinia, cowpox or ectromelia viruses. Type specific differences result largely from extensive, near terminal variations in length and sequence.

Representative Orthopoxvirus genomes have rapidly renaturing terminal restriction fragments confirming the presence of near terminal, covalent cross-links. Terminal restriction fragments from the same or different genomes generally cross hybridize indicating the presence of near terminal repetitions of mol. wt. up to 6 × 106 and which share at least a subset of common sequences. Variola strains however, appear to lack such sequences from one specific terminus which maps shorter than that of related viruses.

A search was made in LCM virus-immune mice for virus-specific antibodies. With the help of an L-cell plaque assay, neutralizing antibody was readily detected. There were no essential differences between mouse strains, but marked differences existed between virus strains. Whereas the inoculation of either large or small doses of WE strain virus led to the early production of considerable concentrations of neutralizing antibody, in the case of E-350 strain virus, high doses were required and a much longer time interval had to elapse before the threshold of detection was attained. In addition to neutralizing antibody, LCM virus-infected mice produced sensitizing antibody (detected by the enhancing effect of an anti-mouse Ig anti-serum on the ability of a serum to reduce virus infectivity) and complement-fixing antibody. Previous failures to detect neutralizing antibody in LCM virus-immune mice might have been caused by properties of the chosen virus, but in many instances lack of a suitable assay host is a more likely explanation.

Bone marrow of leukaemic patients, non-leukaemic patients and normal individuals were co-cultivated with the canine cell line A7573. These co-cultures were screened for retrovirus antigens by means of the indirect cytoplasmic immuno-fluorescence assay (IFA). Rabbit antisera directed against the major structural protein (P30) of woolly monkey (simian) sarcoma leukaemia virus (grown in human lymphoid cells) and Rauscher murine leukaemia virus were used for testing.

After 2 months in culture, 6 of 17 co-cultures containing cells from leukaemic patients showed positive staining in the IFA with the anti-simian virus serum. In control dog cells fluorescence was never observed. Five of the six positive cultures were derived from leukaemic children. One of 12 co-cultures of the non-leukaemic group and one of nine normal bone marrow co-cultures were positive with the simian virus antiserum. None of the 38 co-cultures stained positive in the IFA with Rauscher virus antiserum.

Absorption of the simian virus antiserum with calf serum or mouse mammary tumour virus had no dramatic effect in the IFA on positive control cells or on cells of a positive co-culture. However, absorption with purified simian virus (grown in rat cells) completely abolished these fluorescence reactions. The results provide evidence that simian sarcoma-leukaemia virus related information was present in the original bone marrow samples and that co-cultivation with permissive mammalian cells enabled the detection of virus footprints.