- Volume 30, Issue 2, 1976

Sindbis virus strain AR339 induces interferon by 3 h after infection at 39 °C and by 8 h after infection at 30 °C. The time course of interferon induction between 4 and 9 h after infection at the restrictive temperature (39 °C) was measured for 24 temperature-sensitive (ts) mutants, all of which induced interferon by 16 h after infection. Mutants showing total RNA synthesis at 39 °C greater than 10% of the wild-type level induced interferon with kinetics similar to the wild-type. Of those mutants showing 1 to 10% of the wild-type level of RNA synthesis at 39 °C, four induced interferon with kinetics similar to the wild type, whereas six showed a lag in induction. Nine mutants, showing 0 to 5% of the wild-type level of RNA synthesis at 39 °C, and the wild type, were examined for single and double-stranded RNA synthesis at 30 and 39 °C, using a combination of lithium chloride precipitation and CF11 cellulose chromatography. Six of these mutants showed a lag in interferon induction at 30 °C, while three showed no lag. For all nine mutants, double-stranded RNA synthesis at 39 °C was undetectable, although easily detectable for the wild type. Both the wild type and the mutants synthesized double-stranded RNA at 30 °C. For all mutants, time of interferon induction at 39 °C was correlated with c.p.e. as measured by trypan blue uptake 30 h after infection.

The mutant F104, showing undetectable RNA synthesis and a long lag in interferon induction at 39 °C, was examined for interferon to be induced with wild-type kinetics, and this was correlated with an increased c.p.e. 30 h after infection. Increased RNA synthesis and infectious virus production were detectable at 30 °C after an initial hour at 30 °C.

It is concluded that, for interferon to be induced with normal kinetics, early virus functions are required, but that normal levels of double-stranded RNA synthesis are not necessary. The events which lead to interferon induction also lead to visible c.p.e.

The effect of human cytomegalovirus (CMV) on cell DNA synthesis and mitotic activity in hamster embryo fibroblasts was examined. The results indicated that CMV infected cells had increased rates of cell DNA replication and mitotic activity. Detection of the effect of CMV on these two parameters necessitated arrest of cells prior to infection with low serum concentrations. This lowered the background levels of DNA synthesis and cell division so that the effect of virus infection could be detected. The data indicate that cells arrested prior to infection demonstrate increased susceptibility to virus infection. It was also observed that the effect of CMV on both DNA replication and mitotic activity could be enhanced by irradiation with ultraviolet light of the virus prior to infection.

Thirteen ts mutants of type 2 herpes simplex virus were backcrossed to a syncytial but not temperature-sensitive mutant of wild-type virus. This was an attempt to introduce a third marker, syncytial plaque morphology or syn, into at least some of the ts mutants. Three ts mutants carrying the syn marker were obtained but only one, ts 9, was satisfactory for genetic experiments. Three-factor crosses were carried out between ts 9 syn and the mutants which determined the order of eleven ts mutations relative to both the ts 9 mutation and the syn mutation. A provisional linkage map based both on the order derived from the three-factor crosses and on map distances from recombination frequencies has been prepared: it contains nine ts mutations and the syn mutation.

Controlled disruption of 60S AMV RNA with formamide was used to prepare 50–55S and 30–40S RNAs. When the activities of these RNAs as templates for AMV reverse transcriptase were compared it was found that 50–55S RNA was 1.5 times and 30–40S RNA 2 to 3 times more active than 60S RNA. The 30–40S RNA produced by heating, instead of formamide disruption, was inactive as a template but activity was restored by addition of oligo(dT). 40% of the 4S RNA initially associated with the 60S RNA remained associated with all the RNA species obtained by formamide treatment but was lost on heating. It is concluded that this RNA acts as resident primer whereas the other 60% of the 4S RNA is less firmly bound and appears to have little or no primer activity. Removal of the less firmly bound 4S RNA increases the template activity of the viral RNA.

Organotypic cultures of newborn hamster choroid plexus were inoculated with equal titre doses of newly isolated or hamster adapted strains of mumps virus. The ultrastructure of virus replication in choroid epithelial cells of the cultures was compared. No qualitative differences were observed; however, the adapted strain produced significantly greater numbers of virions and earlier destruction of the cultures. These findings are consistent with previous in vivo observations of the ultrastructure of the replication of these strains in the newborn hamster central nervous system. This in vitro study lends further support to the hypothesis that differences in the in vivo biological effects of the virus strains are primarily the result of virus-cell rather than virus-host interactions.

Three new complementation groups of type 2 herpes simplex virus are described bringing the total number of complementation groups characterized to 13. Of the three new groups, ts 11 fails to make virus DNA at non-permissive temperature (38 °C) whereas ts 12 and ts 13 synthesize only very small amounts of virus or cellular DNA at 38 °C. ts 11, like ts 9 (Halliburton & Timbury, 1973) fails to switch off host cell DNA synthesis at 38 °C. That this is a failure to switch off cell DNA rather than a stimulation of cell DNA synthesis was confirmed in experiments using resting cells. Both the inability to make virus DNA and the inability to switch off cell DNA are reversed in temperature shift-down experiments with cells infected with ts 9 or ts 11. In temperature shift-up experiments, cellular DNA synthesis is inhibited after the shift but virus DNA is only made in very small amounts, probably due to the continuing functioning of a protein made at permissive temperature (31 °C) before the shift but which cannot be made at 38 °C. The shift-down experiments and the fact that ts 9 and ts 11 complement one another, suggest that the switch-off of host cell DNA synthesis may involve more than one virus specified function. U.v. irradiated virus fails to switch off host cell DNA synthesis.

The resistance of mice to intraperitoneal and intramuscular infection with fixed rabies virus increases with age. Treatment of mature animals with either silica, indian ink or antimacrophage serum, which are cytotoxic for macrophages, reduced their resistance to intraperitoneal, but not to intramuscular or intracerebral infection. Transfer of peritoneal macrophages from adults to syngeneic suckling mice delayed but did not prevent mortality from intraperitoneal infection: transfer of peritoneal macrophages to intramuscular sites of infection did not protect adult mice.

Rabies virus was phagocytosed by peritoneal macrophages in culture but neither replicated nor induced interferon. Evidence of active intracellular destruction of virus was obscured by thermal inactivation at 37 °C. Less inactivation occurred at 33 °C. Infected macrophages from suckling mice, but not those from adult mice, spread infection to susceptible cells.

Incubation of adenovirus type 2 infected cells at 42 °C resulted in an inhibition of assembly of virus particles although all the major viral structural polypeptides and virus-induced cellular polypeptides so far identified were detected by electrophoretic analysis. Selective high salt-acid-urea extraction of low mol. wt. polypeptides revealed the absence of protein VII at 42 °C whereas precursor polypeptide P-VII and core protein V were found. Pulse-chase and temperature shift experiments indicated that cleavage of P-VII into VII was a reversible thermosensitive process, requiring de novo protein synthesis after shift-down to 37 °C. Virus particles assembled at 37 °C after transfer from 42 to 37 °C contained both viral DNA and polypeptides pre-labelled during the eclipse phase at 42 °C, including core protein VII.

The mol. wt. and molar ratios of the Hind III and Hpa I fragments of HSV-1 DNA and the Eco RI fragments of HSV-2 DNA have been determined. Results obtained suggest that DNA isolated from both HSV-1 and HSV-2 consists of molecules with four different sequence arrangements which are present in similar amounts. Our explanation of the cleavage patterns of these four genome arrangements with the different restriction enzymes is presented.

Some of the possible implications of these four genome arrangements for genetic recombination are discussed.

Infection of human embryonic fibroblast cell monolayers with neutral red and light-inactivated herpes simplex virus, type 2 (HSV-2) at supra-optimal temperature (42 °C) resulted in persistence of viable cells in suspension culture at 37 °C which have properties in common with virus transformed cells: formation of cell aggregates, HSV-2-specific antigens and colony formation in soft methyl cellulose medium. These data are consistent with the idea that photodynamic inactivated HSV-2 has potential oncogenic activity.

The polyadenylation of rhinovirus-specified RNA in aneuploid HeLa cells and diploid human embryo lung (HEL) cells has been investigated. Polyadenylation occurs at a faster rate and more extensively to virus-specified RNA in infected HEL cells compared with HeLa cells. Poly(A) is added post-transcriptively in both cell types. Furthermore the addition of adenosine or poly(A) during virus multiplication increases the polyadenylation of virus single-stranded RNAs in both cell types, but to a greater extent in HeLa cells.

Immune complexes could be formed with hepatitis B Ag and anti-IgG serum after treatment of the antigen with detergent, CsCl or glycerol, but not before. It is suggested that an antigenic determinant specifically reacting with anti-IgG forms an integral part of hepatitis B Ag. This determinant is ordinarily obscured in the undamaged antigen.