- Volume 6, Issue 2, 1970

P 2 phage particles contained DNA (38 %) and protein (62 %), and were assigned a particle weight of 5·8 × 107 daltons, based on the known molecular weight of P2 DNA. The extinction (1 cm.) of suspensions of 1011 particles per ml. was 0·09 at 260 nm. wavelength. In purified preparations 20 to 50 % of the particles present were not detected by biological assay. The preparations were heterogeneous in respect to heat stability. Even at relatively low temperatures, changes in light scattering accompanied heat inactivation.

The physiological defects associated with 17 temperature-sensitive mutants of the kenya 3/57 strain of foot-and-mouth disease virus were examined in terms of the ability of the mutants to produce infective RNA and complementfixing antigen under restrictive conditions. The yields of infective RNA obtained with different mutants ranged from less than 0·31 to 87% of normal, and of complement-fixing antigen from 12·6 to 99·6 % of normal. Genetic recombination was observed in crosses of mutants with different physiological effects, such that the mutants could be arranged in five groups.

The ihd strain of vaccinia virus caused the rounding of all LLC-MK2 cells in a culture within 2 to 4 hr after infection. One µg./ml. of either cycloheximide or streptovitacin A protected 75 % of the infected cells from early cytopathic effects occurring 4 hr after infection and treatment. As much as 83 µg./ml. of puromycin was required to achieve the same degree of inhibition. Puromycin (330 µg./ml.) continued to protect > 90 % of the infected cells 24 hr after infection, while < 40 % of cells treated with cycloheximide (300 µg./ml.) or streptovitacin A (300 µg./ml.) were protected from virus cytopathic effects at 24 hr. Seventy-five per cent inhibition of virus reproduction was achieved with 24µg./ml. puromycin, 0·125 µg./ml. cycloheximide and 0·062µg./ml. of streptovitacin A. When either puromycin (330 µg./ml.) or cycloheximide (300 µg./ml.) was removed from infected cultures after a treatment period of 4 hr, cell rounding began shortly thereafter and was complete 1 hr later. However, after the removal of streptovitacin A (300 µg./ml.) there was a 1 hr delay before virus cytopathic effects appeared. The cytopathic effects reached maximum 3 hr after the removal of the compound. When streptovitacin A was removed from LLC-MK2 cells infected with vaccinia virus the yield was 30 % of control 24 hr after infection. Virus yields from L2 cells treated with streptovitacin A, however, were only 1 to 5 % of control yields. The aminonucleoside of puromycin (330 µg./ml.) markedly inhibited virus replication, but did not significantly alter the development of virus cytopathic effects.

These findings are taken as further evidence that the early cytopathological effects induced by vaccinia virus result from the synthesis of virus-induced protein(s).

The three species of virus RNA (45 s, 26 s and 20 s) formed during infection of chick embryo cells with Semliki Forest virus were isolated by sequential extraction of the cells with phenol and then with phenol + sodium dodecyl sulphate. They were then separated either by sucrose density gradient centrifugation, or by polyacrylamide gel electrophoresis. The 20 s RNA was relatively resistant to the action of ribonuclease, in contrast to the single-stranded 26 s and 45 s species. The 20 s RNA was the first species to be formed in infected cells followed by 26s and then by 45s RNA. An attempt to demonstrate the intermediate role of 20s RNA in virus RNA synthesis by the displacement of radioactive by non-radioactive uridine (‘pulse-chase’ experiment) was unsuccessful, but both 26 s and 45 s RNA displaced radioactivity from 20 s RNA on heating above the thermal transition temperature and slow cooling. Comparison of the sedimentation velocities and electrophoretic mobilities of the 26s and 45s RNA species, together with the sharp thermal transition at 65° of 45s RNA to 26s RNA suggested that 45s RNA consisted of two 26s RNA species, and that the different sedimentation characteristics did not indicate different conformations of the same molecule.

The fate of [14C]RNA of fowl plague virus was studied in unirradiated and u.v.-irradiated cultures of chick embryo fibroblasts. U.v.-irradiation of cells did not prevent virus penetration into the cell and deproteinization of virus RNA and its penetration into the cell nucleus. However, RNA penetrating into nuclei of irradiated cells underwent practically complete degradation. RNase activity increased two- to threefold in the nuclear fraction of irradiated cells. This RNase may participate in the process of degradation of virus RNA in nuclei of irradiated cells.

Semliki Forest virus in the crude supernatant from a culture of chick embryo cells was specifically adsorbed by a disulphide-linked immunosorbent prepared from the γ-globulins of rabbits immunized by Semliki Forest virus grown in mouse brain. Two to 6 ml. of rabbit antiserum of neutralization index 4 to 5 yielded 10 mg. of immunosorbent which bound 1 to 6 × 1010 p.f.u. from 2 to 30 ml. of culture supernatant. Virus, free from most chick cell constituents, could be eluted from the immunosorbents by treatment with bicarbonate/carbonate buffer at pH ii-i. The immunosorbents could be cycled at least five times and about 50 % of the adsorbed virus infectivity recovered at each cycle after the first, from which only 10 to 20 % was recovered. In capacity experiments, immunosorbents derived from chicken antisera against Semliki Forest virus grown in chick cells showed no advantages over those derived from rabbit antisera.

Mature particles and the early stages of growth of tomato spotted wilt virus (TSWV) were examined by electron microscopy in thin sections and by negative staining. Early evidence of infection included: (a) amorphous, darkly stained material in the cytoplasm; (b) spherical virus-like particles 100 nm. in diameter, with two concentric membranes; (c) configurations interpreted as membranes budding to form the particles in (b); (d) mature virus particles resembling the inner membranes plus cores of the particles in (b). Similar structures were seen in thin sections of developing local lesions. Local and systemic infections more than five days old contained only mature particles, and not structures (a), (b) and (c).

A possible sequence for virus maturation is suggested. The evidence does not support the suggestion that TSWV is a myxovirus.

The optimal conditions for an immunofluorescence assay in HEp2 cells of a strain of foamy virus were investigated. Adsorption for 2 hr was followed by incubation at 37° for 65 hr. The validity of the assay was established and it was shown to be as sensitive as titration of cytopathic effect at limit dilution. The sensitivities of BHK 21, HEp 2, RK 13 and vervet cells to foamy virus were compared and distribution of virus antigen in HEp 2 cells at various stages of infection were investigated.

A precise, reproducible, and sensitive serum neutralization test was developed to estimate Venezuelan equine encephalomyelitis neutralizing antibody activity within 24 hr. The test depends on the interactions of virus with antiviral globulins and of the resultant complexes with anti-gamma globulin (IgG) antibodies. The 50 % serum neutralizing dilutions were calculated from the reduction of fluorescent cells in McCoy cell monolayers resulting from the neutralization of infective virus by antibody. In comparative estimates of the neutralizing activity of human and monkey antisera, the sensitivity of the serum neutralization anti-IgG test was several hundredfold greater than that of the conventional serum neutralization test in mice.

In HeLa cells infected with poliovirus, shifts from optimal to supraoptimal temperatures in the middle of the infectious cycle resulted in extensive degradation of newly made virus RNA. This temperature-induced degradation was not observed until about 3 hr after infection. It did not require simultaneous synthesis of proteins. During the first 3 hr of infection at 39·5°, RNA of temperature-resistant poliovirus strains replicated much faster than at 36°. Subsequently, degradation became predominant. Thus, temperature-resistant strains partially escaped the effects of degradation by faster replication. During the first 150 min. approximately, the RNA of temperature-sensitive strains replicated as well at supraoptimal as at optimal temperatures. Subsequently, newly made virus RNA was degraded. The inhibitory effect of supraoptimal temperatures may be due to release, or activation, of a ribonuclease.

Erythrocytes from various species showed a gradient of sensitivity to agglutination by arboviruses, from reptile erythrocytes, relatively insensitive, to avian erythrocytes, the most sensitive tested. Lipids extracted from both inagglutinable and agglutinable erythrocytes inhibited arbovirus haemagglutination. Lipid fractionated on DEAE-cellulose columns yielded two major inhibitory fractions, one containing only neutral lipid including cholesterol and another phospholipid and neutral lipid but no cholesterol. Fractionation of the lipid on silicic-acid columns resulted in a loss of activity in the fractions compared with the total lipid extract. Recombination of the fractions did not restore the inhibitory activity. Similar loss of activity followed treatment of lipid extracts with digitonin.

No evidence of an essential component in inhibitory lipid mixtures was observed. Tests of mixtures of standard lipids gave results suggesting the need for suitable arrangements of lipids in aqueous solutions for interaction with arboviruses. Only certain combinations of lipids were potent inhibitors. The interactions observed were thought to be between arboviruses, which contain lipid, and artificial lipid aggregates in aqueous solutions. The arrangements of lipids were not thought analogous to receptor sites on erythrocytes.