- Volume 65, Issue 6, 1984

Two temperate bacteriophages have been isolated from Bacillus larvae: PBL1 and PBL0.5. Strains lysogenic for either of these phages are immune to lysis by the same phage but are sensitive to the other phage. PBL1 has an oval head, a non-contractile tail, and a base plate with a pin structure but no apparent tail fibres. The genome of PBL1 consists of double-stranded DNA with a molecular weight of 24.1 (±0.6) × 106, a G + C content (derived from melting temperature) of 41.5%, and cohesive ends. Restriction enzyme analysis permitted construction of a physical map of the genome.

A pseudo-crystalline array of subunits has been observed on particles of the La Sota, in contrast to the Ulster, strain of Newcastle disease virus (NDV) grown in MDBK tissue culture without trypsin. This regular arrangement of subunits was associated with the semi-permissive nature of the tissue culture system, as it disappeared when trypsin, which allows infectious virus to be made, was added. The phenomenon described was considered to be related to the crystalline array of matrix protein which has been described inside the envelope of Sendai virus and NDV by others.

The effects of monensin on cells infected with pseudorabies virus have been studied. Treatment with monensin (1 µm) prevented the secretion of the 89K pseudorabies virus-induced sulphated glycoprotein. A polypeptide with a mol. wt. of 78K was secreted into the medium from monensin-treated BSC-1 cells. Mannose labelling showed that initial glycosylation proceeded normally in the presence of monensin, although the absence of fucose label revealed that the later stages in processing were blocked. No intracellular accumulation of virus-induced proteins was found in monensin-treated cells. Infectivity assays, measurement of thymidine-labelled particles and electron microscopy showed that monensin blocked the release of virus particles from cells, although viral morphogenesis was unaffected.

Embryonal carcinoma (EC) cell lines established from human testicular germ-cell tumours produce, at low frequency, virions morphologically identical to type C retroviruses that have been observed by other workers in human placental tissues. The virus particles are formed while budding from the cell surface, and their numbers are increased by inducing the EC cells with 5-iodo-2′-deoxyuridine and dexamethasone. Assays for RNA-dependent DNA polymerase (reverse transcriptase) associated with purified virions suggested a low level of activity. In addition, another type of virus is occasionally produced by induced cells of three EC lines. These particles also form during the process of budding from the cell surface, but they have surface projections (spikes). Extracellular spiked virions frequently are pleomorphic, with a condensed, eccentric nucleoid, and thus morphologically resemble type B retroviruses. No virions of either type were detected with or without induction in cultures of differentiated EC cells or in cultures of yolk sac carcinoma or teratoma cells, both of which are considered malignant but differentiated derivatives of EC cells. The lack of virion production by these differentiated cells suggests developmental regulation of virus replication.

We provide evidence that (i) variants lacking individual herpes simplex virus type 1 (HSV-1) XbaI sites can be selected following extensive XbaI treatment of the viral DNA and can be recombined to produce HSV-1 variants lacking two of the four sites normally found, (ii) all XbaI sites can be removed from a viable intertypic recombinant HSV genome, (iii) following XbaI treatment, different mutants with deletions (0.15 to 8.8 kb) in the long repeat (TRL or IRL) and long unique regions can be readily isolated, as well as mutants with novel XbaI sites, (iv) several mutants with deletions in one of the repeats (TRL or IRL) have a measurable growth disadvantage in tissue culture.

Treatment of HeLa cells with human lymphoblastoid interferon [HuIFN-α(Ly)] induced an antiviral state that rendered the cells refractory to infection by herpes simplex virus type 1 (HSV-1), and the yield of infectious HSV-1 was reduced by 98%. Analysis of the mechanism of the anti-herpes action of IFN indicated that no gross inhibition of viral protein synthesis took place and late proteins appeared, although the synthesis of some of them was partially inhibited. No differences were apparent between the glycoproteins or phosphoproteins synthesized in control and IFN-treated HSV-1-infected HeLa cells. No inhibition of the formation of new virus particles from IFN-treated cells was evident as assessed by electron microscopy and biochemical analyses, although their infectivity was drastically reduced. These virions exhibited some differences in their content of a few proteins as determined by PAGE. Studies on the second infection cycle with virions obtained from IFN-treated cells suggested that they attached and penetrated HeLa cells but that their development was impaired, since no viral protein synthesis was observed during the second infection. Altogether these results do not support the idea that the molecular mechanism of action of human IFN against HSV-1 is mediated via the 2′–5′A system, or by the inactivation of initiation factors. Rather, they focus the problem of the anti-herpes action of IFN at the level of formation of defective virions.

With the aim of isolating temperature-sensitive (ts) mutants defective in virus maturation or glycoprotein transport, Uukuniemi virus, a bunyavirus, was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. Out of 13 initial clones unable to grow at 39 °C (non-permissive temperature), five mutants which grew to titres above 107 p.f.u./ml at 32 °C (permissive temperature) were selected for further studies. The mutants fell into two coinciding recombination-complementation groups. Three group I mutants (ts7, 8 and 12) and two group II mutants (ts6 and 11) synthesized all three RNA segments and were able to form the corresponding nucleoproteins at 39 °C. Thus, members of these two recombination groups had a RNA-positive phenotype. All five mutants showed immunofluorescence when cells were stained at 39 °C using a double-staining technique employing monoclonal antibodies against the glycoproteins G1 or G2, and polyclonal antibodies against the nucleoprotein, N. We have previously shown that in cells infected with wild-type virus both the G1/G2 and the N proteins accumulate in the Golgi complex, the site of virus maturation. In cells infected with ts12, accumulation of G1 and G2, but not N protein, was observed in the Golgi complex at 39 °C. The N protein was found evenly scattered in the cytoplasm, suggesting lack of interaction between the G1/G2 and N proteins. With ts6 and 11, G1 and G2 appeared to accumulate and aggregate in the endoplasmic reticulum (ER) at 39 °C. The location of the N protein coincided with that of the aggregated glycoproteins, suggesting that the N protein interacted with G1/G2 already in the ER. Thus, these mutants may prove valuable tools in studying the mechanism of Uukuniemi virus maturation.

The suggestion that antibodies might enhance rabies virus infection of macrophages through opsonization of immune complexes was tested in vitro by adaptation of the rapid fluorescent focus inhibition technique for the examination of a macrophage cell line (P388D1). Some enhancement of rabies virus infection was shown. The relationship between such enhancement with the ‘early death’ phenomenon and its occurrence in vivo is discussed.

This study was initiated to determine whether the Theiler’s murine encephalomyelitis viruses (TMEV) use the same strategy of gene expression as other picornaviruses. The precursor-product relationships apparent from pulse-chase experiments indicated that the virus-specified polypeptides of the GDVII strain are generated by post-translational cleavages. Pactamycin mapping experiments also showed a gene order for GDVII virus similar to that derived for other picornaviruses by this technique. Finally, six other TMEV induced species of polypeptides similar to those of GDVII virus, but there were minor differences in some of their electrophoretic mobilities.

Pretreatment of HeLa cells with human interferon (IFN) resulted in the inhibition of herpes simplex virus (HSV) replication. We examined the stages in the replication of HSV type 1 and type 2 that were affected by IFN. The rate of synthesis of the HSV immediate-early (α) proteins was inhibited in IFN-pretreated HeLa cells. The subsequent inductions of HSV early (β) genes, determined by measuring the levels of cytoplasmic mRNA specific for the thymidine kinase, as well as the DNA polymerase enzyme activity, were also suppressed in the IFN-pretreated cells. These results indicate that IFN inhibits HSV replication primarily at a very early step, either prior to, or during the synthesis of α-proteins.

F-IFr cells, which are more resistant to the anticellular and antiviral action of human α and β interferons (IFN-α and IFN-β) than the parental RSa cells, were also found to be more resistant to both the anticellular and antiviral effect of IFN-γ. A high level of 2–5A synthetase was induced by treatment with IFN-α or -β, but induction of 2–5A synthetase was not detected after IFN-γ treatment of the cells. F-IFr cells had less than half the number of specific binding sites for IFN-α than the parental RSa cells.

There was strong interference between various virulent and avirulent strains of Semliki Forest virus (SFV) and their respective defective interfering (DI) viruses but in other combinations interference was variable: it could be equally strong, weak or could not be demonstrated. On passage, this spectrum of interfering activity changed, some combinations showing greater interference than before and others less. Heterotypic interference between DI SFV, DI Sindbis virus and standard viruses was clearly demonstrated although this was strongest between DI SFV preparations and Sindbis standard virus than in the reciprocal combinations. Variation in interference between DI SFVs and different SFV strains was similar in magnitude to that between DI SFVs and Sindbis virus, suggesting that a similar DI RNA sequence is recognized by both viruses.

Evidence is presented for the first time that a human candidate calicivirus (HCV) replicates in human embryo kidney cells when trypsin is incorporated in the culture medium. The virus multiplies in the presence of actinomycin D and radiolabelling experiments with [3H]uridine indicate that it has an RNA genome. These observations provide further support for the view that HCV should be tentatively classified as a member of the Caliciviridae.

We previously reported that nucleoproteins (NPs) of human influenza viruses are cleaved in infected cells and, as a result, two forms of NP, uncleaved (mol. wt. 56000) and cleaved (mol. wt. 53000) were accumulated late in infection. Here, we report that NPs of animal influenza viruses of non-human origin (isolated from pigs, equids, seals, whales, birds) exhibited proteolytic resistance in infected cells and did not undergo a change in mol. wt. in the course of infection. The resistance of the animal virus NPs to proteolytic cleavage was shown to be a virus-specific property and not the consequence of a low level of proteolysis in infected cells. Influenza A/H3N2 viruses isolated from swine in Hong Kong in 1976 were found to have a cleavable NP like that of ‘human’ viruses, supporting the hypothesis concerning the ‘human’ origin of these strains. The NP of human influenza virus (A/Aichi/2/68) adapted to an animal host (mouse) retained susceptibility to limited intracellular proteolysis. Thus, NP resistance to cleavage seems to be a stable viral characteristic enabling the NP56→NP53 modification to be used as an indication of the origin of influenza viruses.

Na-K changes and cytotoxic effects were monitored in HeLa cells infected with encephalomyocarditis (EMC) virus, human rhinovirus or vesicular stomatitis virus (VSV). Whereas in all three cases Na-K changes followed viral inhibition of cellular protein synthesis, such changes preceded trypan blue staining of rhinovirus-infected or VSV-infected cells and paralleled trypan blue staining of EMC virus-infected cells. In each case a progressive reduction in cellular electrical volume was associated with Na-K changes. K leakage from EMC virus- or rhinovirus-infected cells clearly exceeded Na gain by these cells. These findings are basically similar to those previously reported for poliovirus-infected HeLa cells. It is concluded that Na-K changes induced by picornaviruses or VSV do not trigger shut-off of HeLa cell protein synthesis, but may contribute to late manifestations of cytotoxicity.