- Volume 27, Issue 1, 1975

The fate of parental SV40 DNA in monkey kidney cells was investigated by infecting the cells with purified virus labelled with [3H]-thymidine. 5-Bromodeoxyuridine (200 µg/ml) was added to the cells at 2 h after infection to label virus progeny DNA. At 72 h post-infection the cells were harvested and virus DNA was extracted and fractionated by isopycnic sedimentation in CsCl solution. The following DNAs with characteristic densities were found: light (LL), 1.70 g/ml; hybrid (HL), 1.75 g/ml and heavy DNA (HH), 1.80 g/ml.

Of the total cell-associated [3H]-radioactivity (derived from parental virus), more than 90% was recovered in unreplicated parental DNA (LL DNA), about 2% was recovered in the HL DNA and about 0.7% was associated with the HH DNA. The unreplicated parental DNA was present as uncoated intact DNA complexed with proteins present in the infected cell. The HL DNA contains one light parental and one heavy progeny DNA strand. The nature of the radioactivity present in the HH DNA remains to be determined.

Infection of LS cells with HSV-1 resulted in an inhibition of spermidine and spermine synthesis from putrescine, possibly through inhibition of host cell protein synthesis. The rate of putrescine uptake increased soon after infection, and later, polyamines were lost from the cells. Inhibition of spermidine and spermine synthesis by methylglyoxal bis(amidinohydrazone) did not affect virus replication.

Interferon-treated L cells exhibit a specific enhanced susceptibility to the cytotoxic and interferon inducing activities of double-stranded RNAs such as poly(I).poly(C). These activities remained closely linked through widely varying assay conditions, involving, for example, different time and dosage schedules of poly(I).poly(C), suggesting that there is at least one common step in the mechanisms leading to interferon formation and toxicity in interferon-primed cells exposed to poly(I).poly(C). However, some procedures such as addition of metabolic inhibitors (actinomycin D, cycloheximide) and repeated administration of poly(I).poly(C) suppressed the interferon inducing capacity of poly(I).poly(C) without a concomitant decrease of toxicity. Other procedures such as brief treatment of the cells with interferon or DEAE-dextran permitted full expression of the interferon inducing activity of poly(I).poly(C) without any sign of toxicity. The latter results suggest that the mechanisms underlying interferon production and toxicity of poly(I).poly(C) in interferon-treated L cells diverge from a certain point onward.

The proportion of cells absorbing trypan blue (tb + character) can be used to measure the late c.p.e. of wild-type poliovirus (ts +.tb +), which was the same at restrictive (39.2 to 39.6 °C) or permissive (37 °C) temperatures. Of twenty ts mutants, seven showed normal c.p.e. at 37 °C but were defective in c.p.e. (tb) at 39.5 °C; all seven tb mutants have previously been shown (Cooper et al. 1971) to give evidence of a primary defect in replicase I activity (to make the complementary or minus strand of virus RNA). The remainder (tb +) have all previously been shown to give evidence of a primary defect either in replicase II activity (to make progeny plus strands) or in structural protein. Thus, the late c.p.e. is dependent on a product of the replicase I gene, of which the in vivo effector is probably double-stranded RNA. Late c.p.e. is not caused by prevention of host protein, RNA or DNA synthesis and is not necessarily correlated with lysosomal enzyme release. The tb mutants were also defective in inducing early changes in chromatin (chr) and in prevention of thymidine incorporation (pti), but the tb and pti/chr characters are probably independent expressions of replicase I activity. Virus growth does not depend on repression of DNA synthesis. Poliovirus represses the activities of host DNA-dependent RNA polymerase I and II to an equal extent. There is no evidence that repression of DNA or RNA synthesis results from direct interaction of a virus protein with the DNA.

A number of temperature-sensitive mutants were isolated from two strains of foot-and-mouth disease virus (FMDV). Various properties of the mutants were examined including comparative growth curves at permissive and restrictive temperatures, cut-off temperatures, thermal lability and pH sensitivity. Recombination was observed between various pairs of mutants of FMDV strain Pacheco. It occurred early in the growth cycle and the proportion of recombinants remained constant thereafter. Maximum recombination was achieved if the input multiplicity of each virus was 6 p.f.u./cell or greater, provided the ratio of the input multiplicities did not vary by more than a factor of two. Day-to-day variations could be substantially reduced by normalizing recombination frequencies in terms of a standard cross. The results suggested that genetic mapping was possible with two- or three-factor crosses.

After exposure of human embryonic fibroblasts to BK virus, virus particles adsorbed to the plasma membrane were engulfed by pinocytosis or captured by vesicles, possibly originating from the endoplasmic reticulum, within 2 h after infection. Most of the virus particles were then transported into lysosomes or into the nucleus, while a small amount of virus was found free in the cytoplasm. Virus particles entered the nucleus between 2 and 12 h after infection, were still detectable in the nucleus at 24 h after infection and became morphologically undiscernible at 30 h after infection, suggesting that a nuclear uncoating mechanism was active between 24 and 30 h after infection. Virus progeny started to appear in the nucleus of infected cells at 4 days after infection, but not until 7 to 8 days after infection did the virus escape into the cytoplasm and cell degeneration became evident. The possible explanations for the long replicative cycle of BK virus are discussed.

Hepatitis B surface antigen was adsorbed to insolubilized sialic acid-specific haemagglutinin isolated from the haemolymph of Limulus polyphemus. Treatment of the antigen with Vibrio cholerae neuraminidase (EC 3.2.1.18) resulted in the release of sialic acid and in an increase of the isoelectric point from pH 4.35 (for subtype ad) or 4.9 (for subtype ay) to pH 5.45. Treated, but not untreated, antigen incorporated [14C]-sialic acid when incubated at 37 °C with sialyl transferase (EC 2.4.99.1) and cytidine-5’-monophosphate-[14C]-sialic acid. The major portion of [14C]-sialic acid was linked to a glycoprotein with an apparent mol. wt. of 26 × 103. De-sialylated antigen had a drastically reduced in vivo life span in rabbit plasma and elicited a higher humoral antibody response than intact antigen (subtype ad). Antigen-stimulated proliferation of lymphocytes, measured 3 months after immunization, was observed only with cells from rabbits injected with neuraminidase-treated antigen.

Intraperitoneal injection into mice of BHK-HVJ cells (BHK cells persistently infected with HVJ) induced considerable amounts of circulating interferon. Sonication of BHK-HVJ cells almost totally abolished their interferon inducing capacities. This result suggests that a certain native organized structure of virus infected cells might be essential for production of interferon in this system, as is suggested in the in vitro system.

An Aedes albopictus cell line was found contaminated with structures morphologically compatible with an alphavirus. Rapid isolation of a cytopathic virus was effected by combining sonication, concentration with Aquacide IIR, rate zonal sedimentation and subsequent plating of fractions on Vero cells under agar overlay. The virus caused neither death nor disease on inoculation into infant and adult mice. It produced a c.p.e. in Vero and BHK 21 cells, and multiplied in Singh's Aedes aegypti cells. The virus was identified serologically as chikungunya by complement-fixation and plaque reduction neutralization test. Virus was not detected in a single attempt by these methods in the American Type Culture Collection A. albopictus line. The presence of chikungunya virus in A. albopictus cells is not easily recognized and may complicate interpretation of experimental results.

Polyacrylamide-SDS gel electrophoresis indicated that cauliflower mosaic virus (CIMV) particles contain two major and possibly one minor structural polypeptides with mol. wt. of 68000, 42000 and 55000, respectively. Up to seven other minor bands produced by disrupted virus were probably degradation products or stable aggregates of the structural polypeptides. The amino acid composition of CIMV protein differs from that of most other plant viruses in containing about 18% lysine. The content of basic amino acids in CIMV protein suggests that at least one of the polypeptides is an internal component with a strong affinity for DNA.

Bone marrow colony-stimulating activity in the culture media of L cell monolayers treated with Newcastle disease virus or polyriboinosinic:polyribocytidylic acid showed an early increase followed by a marked fall of activity when compared with non-induced cultures.

Preliminary experiments were initiated to test the attenuation for mice of chemically induced temperature-sensitive (Ets) mutants of a virulent strain of Eastern encephalitis (E) virus, and the potential of such mutants as live virus vaccines for mice. The reversion frequencies of eight mutants to temperature insensitivity were measured and the defects in their biosynthesis at the nonpermissive temperature were studied. All eight mutants were less virulent for mice, but the extent of avirulence varied with the mutant and route of injection. The mutants selected as potential vaccines protected mice against subsequent challenge at 10 to 21 days by the virulent virus of strain E. Neutralizing antibody activity was detected in almost all of the mutant-infected mice after 10 days, and was found in all infected mice at 21 days. Immunization by two doses of virus induced a very high protection against intracerebral challenge by virus strain E.