- Volume 43, Issue 2, 1979

The phosphorylation of arabinofuranosylthymine (araThd) has been studied both in non-infected cells and in those infected with herpes simplex virus (HSV-1, Lennette; HSV-1, IES and HSV-2, D-316). In these experiments, HSV strains were used which either contain (Lennette, TK+ and D-316 TK+) or lack (IES, TK−) the capacity to induce pyrimidine deoxyribonucleoside kinase. It was found that extracellularly administered araThd is phosphorylated to araTTP via araTMP and araTDP in both non-infected and in HSV-infected cells. The phosphorylating capacity is more than tenfold lower in non-infected cells than in infected cells. Interestingly, cells infected with the TK− strain have a tenfold higher phosphorylating capacity than normal, uninfected cells, a fact which might indicate that host cell deoxythymidine kinase is induced during HSV infection. AraTMP is incorporated into cellular DNA but not into HSV DNA. This finding is in contrast to observations with arabinofuranosyladenine, which is incorporated into both cellular and HSV DNA. In vitro experiments with HSV-induced DNA polymerase show that araTTP strongly inhibits the enzyme activity. Therefore we conclude that the inhibition of HSV DNA polymerase by araTTP (formed intracellularly from araThd) is the explanation for the observed antiviral activity of araThd.

Among the different strains of avian paramyxoviruses isolated from migrating feral ducks, two were identified as Newcastle disease viruses (NDV) and the five others would correspond to a new serotype for which we suggest the name of Duck/Mississippi/75 virus. The characteristics of this new serotype are as follows: (1) Duck/Mississippi/75 virus is able to grow as well in allantoic as in amniotic cavities of embryonated hen′s eggs; (2) the haemagglutinin and haemolytic activities can be detected with hen red blood cells; (3) the neuraminidase hydrolyses the α2 → 3 bonds of the fetuin substrate and its pH activity could be species specific. Antigenically, this serotype is different from all human and animal paramyxoviruses, in spite of an antigenic relationship with NDV.

The genome of a single cell derived from the B16 melanoma contains information for the expression of three distinct biologically active viruses: the N- and B-tropic ecotropic viruses and the xenotropic virus. Their release results in reduction of melanin production. A possible relationship of virus replication to differentiation may be involved.

LLC-MK2 cells chronically infected with two strains of rubella virus, HPV-77 and Thomas, have been examined over several months to find out the mechanism of persistence. Evidence is given for the presence of defective particles in these cultures by finding virion RNA which sedimented at 12S instead of the 40S typical of the fully infectious virus. A ‘provirus’ DNA copy of the rubella virus genome was not detected by methods which included filter hybridization and in situ hybridization, or by treatment of the chronically infected cells with mitomycin C, actinomycin D or 5-bromodeoxyuridine. In addition, the chronically infected cells contained RNA-dependent RNA polymerase activity, but no RNA-dependent DNA polymerase activity.

The Pseudomonas phaseolicola bacteriophage ϕ6 nucleocapsid incorporated ATP, UTP, CTP and GTP into ssRNA products which sedimented at the same rate as heat-denatured small and medium dsRNA components. Hybridization of the separated ssRNA products with denatured dsRNA revealed that the small ssRNA hybridized only to small dsRNA and the medium ssRNA to the medium dsRNA. Synthesis of the large ssRNA was not observed. The RNA polymerase reaction proceeded through a replicative intermediate-like RNA probably via a semiconservative mechanism.

Polyacrylamide gel electrophoresis of bovine rotavirus or neonatal calf diarrhoea virus (NCDV) grown in cell culture resolved eight species of polypeptide. The inner shell particles contained five polypeptides and the outer shell three polypeptides. A major polypeptide of the outer shell was glycosylated. The infectivity of NCDV was enhanced by treatment with trypsin in vitro. All eight polypeptides were affected by trypsin treatment as judged by diminished intensity of polypeptide bands by radiography and several new bands appeared. The intracellular synthesis of NCDV polypeptides was studied by pulse and pulse-chase experiments. Infected cells contained all eight virus capsid proteins and, in addition, three presumably virus-specific polypeptides which were non-capsid polypeptides (NCVP). There was no evidence that any of these polypeptides was processed after synthesis. It is suggested, therefore, that all these polypeptides are primary gene products.

The continuous culture of a hamster melanoma cell line has led to the spontaneous appearance of a retrovirus (HaRV) with typical type-C characteristics. The virus differs from all other known hamster viruses in its ability to transform murine as well as rat and hamster cells with apparent one-hit kinetics. Guinea pig, human and feline cells were not transformed although reverse transcriptase activity was detected in the supernatant from infected human cells. HaRV-transformed hamster embryo cells produced solid tumours (all non-pigmented) in 4 out of 35 animals when injected into hamsters while HaRV-transformed murine cells produced no tumours in mice. Injection of HaRV alone in hamsters, mice and rabbits did not induce tumours. HaRV possesses a 70S RNA which dissociates to 35S in DMSO and has a reverse transcriptase which utilizes the 70S virus RNA as a template. The size, morphology and density (1.15 g/ml) are similar to other known type-C viruses. Polyacrylamide gel electrophoresis indicates the presence of polypeptides analogous to those found in other type-C viruses.

Several properties of an RNA-directed DNA polymerase associated with a hamster retrovirus (HaRV) were examined and found to be similar to other polymerases from mammalian type-C viruses in that the enzyme (i) is more active with Mn2+ than Mg2+, (ii) uses the reverse transcriptase-specific poly(rCm).oligo(dG) template, (iii) possesses substantial endogenous polymerase activity and (iv) is strongly inhibited by homologous antisera and moderately inhibited by antisera directed against other type-C viruses. In contrast to previous reports of polymerases from other hamster viruses, HaRV polymerase is active in endogenous assays and the activity is associated with a 70000 mol. wt. polypeptide in highly purified virions and with 70000 and 85000 mol. wt. polypeptides in fresh, unpurified virus. Only one major peak of polymerase activity eluted from DEAE-cellulose while subsequent elution of this peak from phosphocellulose produced two major peaks of polymerase activity. The mol. wt. of these two peaks were 70000 and 85000 by glycerol density-gradient sedimentation. The HaRV reverse transcriptase and p30 were found to be most closely related antigenically to other rodent retrovirus proteins.

The optimum conditions for infection of turnip protoplasts with turnip rosette virus (TRosV) were 0.2 to 2 × 105 protoplasts/ml, 5 to 10 µg virus/ml, 1 µg poly-l-ornithine/ml in 0.6 m-mannitol, buffered with 5 to 10 mm-tris at pH 7.0 to 8.6. Under these conditions, 70% of protoplasts became infected, as indicated by staining with fluorescent antibody. The proportion of protoplasts infected was determined by factors such as pH and buffer ions and by the concentrations of protoplasts, virus and poly-l-ornithine during inoculation. The period of pre-inoculation incubation of virus and poly-l-ornithine was also critical, but mannitol concentration and the inoculation period and temperature had little effect on percentage protoplasts infected. Time course studies showed a single step multiplication of TRosV; the virus was detected on sucrose gradients at 24 h after inoculation and at 60 h reached its maximum concentration, 3 × 106 virions per infected protoplast.

Three factor crosses were performed between Rous sarcoma virus mutants with temperature-sensitive markers in the pol and src genes and host range markers in the env gene. A number of recombinant viruses appeared to segregate from virus particles which were heterozygous for all three genes under study. The frequency of various recombinant genotypes in the progeny was consistent with there being no greater linkage between the neighbouring gene pairs of pol and env and env and src than between the more distant pol and src. The significance of these results to proposed mechanisms of avian retrovirus recombination is discussed.

RNA oligonucleotide fingerprint analyses indicate that the genome RNA obtained from Trinidad donkey (TRD) Venezuelan equine encephalomyelitis (VEE) virus serotype I A, its vaccine strain derivative TC-83, and the VEE I B virus isolate PTF-39, have almost identical patterns of characteristic ribonuclease T1 resistant oligonucleotides. The TC-83 strain and the I B isolate can, on the basis of these analyses, be considered as variants of the TRD virus and categorized as I AB serotypes. Comparisons made by single and co-electrophoreses of the ribonuclease T1 digests of the RNA species of TC-83 and a VEE I C isolate P676 indicate that 16 of 37 large oligonucleotides of the TC-83 virus co-migrate with the oligonucleotides obtained from the I C isolate. Similar single and co-electrophoreses of ribonuclease T1 digests of the RNA species of TC-83 and a VEE I D isolate 3880 indicate that 18 of 41 TC-83 large oligonucleotides co-migrate with the oligonucleotides obtained from the I D virus isolate. At least nine of the TC-83 large oligonucleotides appear on the basis of these analyses, to be present in the digests of the genome RNA obtained from these selected I B, I C and I D virus isolates. The ribonuclease T1 digests of three I E virus isolates (Mena II, 63U2 and 71U388) give oligonucleotide fingerprints which, although comparable to each other, are more distinct from the I A and I B RNA fingerprints than are those of the I C and I D RNA species. The ribonuclease T1 resistant oligonucleotide fingerprints of VEE virus isolates belonging to serotypes (VEE subtypes) II, III and IV show little similarity to each other or to those of the serotype I virus isolates we have studied. The results obtained here agree with the reported close antigenic relationships of VEE, I A, I B, I C and I D virus isolates, and our studies suggest that these viruses have conserved nucleotide sequences. The I E virus isolates appear to have more distinct nucleotide sequences than do the other serotype 1 viruses. The results also agree with the serological differentiation of VEE, I, II, III and IV subtypes in that the oligonucleotide fingerprints of subtypes II to IV are different from each other and from those of the different serotype I virus isolates. On the basis of antigenic and genome relationships, VEE isolates can be classified as serotypes I to IV with serotype I viruses differentiated into the categories I AB, I C, I D and I E.

Strains of foot-and-mouth disease virus of types O1 and A10 were isolated which showed no significant loss of infectivity upon trypsinization. These ‘trypsin-resistant’ (TR) viruses were obtained by serial passage in BHK cells of virus that was trypsin-treated before inoculation of the cells. Three O1 isolates were cloned and studied further. Cell attachment of those TR O1 variants (OTR1) was not reduced by trypsinization, unlike that of parent virus.

The polypeptide compositions of TR viruses as determined by SDS-polyacrylamide gel electrophoresis were identical with those of parent virus, with the exception of OTR1 which contained an additional polypeptide approx. 3000 daltons larger than VP1. After trypsinization, which normally cleaves VP1, the polypeptide composition of the three TR viruses (including OTR1) and of parent virus did not show any significant difference. In OTR1 both the additional virus protein and VP1 were cleaved into a P18 molecule and smaller fragments. The surface location of this additional polypeptide was confirmed by iodination experiments. It was shown by immunodiffusion experiments that only OTR1 differed from the parent virus. This antigenic change was present on the trypsin-sensitive part of the virus since trypsinized TR viruses (including OTR1) were antigenically identical to trypsinized parent virus.

The electrophoretic mobilities of the three OTR viruses isolated, and of parent virus, differed somewhat before trypsinization. After trypsin-treatment, the mobilities of TR viruses were all increased to the same level; however, their rate of migration was lower than that of trypsin-treated parent virus. This lower mobility of trypsin-treated OTR viruses was the only difference which could be associated with retained infectivity.

An assay for virus antibodies using protein A from Staphylococcus aureus is described. Type B and type C RNA tumour viruses adsorbed on to polystryrene microtitre plate wells were incubated with antiserum and then with 125I-labelled protein A (I-pA) and bound radioactivity was determined. Technical details such as labelling, antigen concentration, storage of I-pA are reported. The specificity of the reaction was investigated in detail by competition experiments with purified unbound homologous viruses. This assay also proved to be sensitive for demonstration of autogenous immunity to both type B and type C RNA tumour viruses. A study using antisera against purified core and envelope virus proteins of mammary tumour and leukaemia viruses suggested that the reaction mainly involves surface antigens of the intact virions.

The stability of the infectivity of Simian rotavirus, SA11, has been analysed and compared to the stability of reovirus type 1. SA11 infectivity was stable to freeze-thawing, sonication, incubation at 25 °C overnight or at 37 °C for 1 h and to treatment with acid, ether, chloroform and Genetron. In contrast to reovirus, the infectivity of SA11 was more rapidly inactivated by heating at 50 °C. SA11 infectivity was inactivated above pH 10.0 and by heating at 50 °C in 2 m-MgCl2, but was stabilized by heating in 2 m-MgSO4; reovirus 1 infectivity was enhanced by heating in MgCl2. Both SA11 and reovirus 1 were inactivated by freezing in MgCl2. These results show that rotaviruses and reoviruses can be distinguished by their patterns of inactivation by physical and chemical agents.

By polyacrylamide gel electrophoresis, Duck/Mississippi/75 virus was shown to contain five different types of polypeptides of mol. wt. ranging from 76 × 103 to 43 × 103, two of which were glycosylated (mol. wt. 76 × 103 and 57 × 103). A comparison of this data with similar results obtained using Yucaipa and Newcastle disease virus (NDV) revealed a similarity with NDV, concerning the number, position and mol. wt. of the polypeptides. Haemagglutinating and neuraminidase properties are associated with surface glycoproteins which represent at least 40% of the virus protein. After KCl and Triton X-100 treatment and centrifugation on linear sucrose density gradients (10 to 25%, w/w) it was possible to isolate glycoprotein VP1 of mol. wt. 76 × 103 with which haemagglutinating and neuraminidase activities were associated.

Fresh mouse scrapie brain and frozen mouse scrapie spleen were homogenized, sized by ultrafiltration to a fraction containing particles of 30 nm to 200 nm and separated by zonal centrifugation in sucrose gradients. In both the brain and spleen, the maximum titre of scrapie infectivity banded between densities of 1.125 to 1.200 g/ml; but in spleen a second fraction of scrapie infectivity was observed between 1.200 to 1.250 g/ml. A unique 30 to 60 nm particle was found in mouse spleen gradient fractions with high scrapie infectivity titres. This particle was not observed in similar fractions isolated from normal mouse spleen or brain and was rarely found in subcellular fractions of low titre from scrapie-infected tissues. The particle was first observed by negative staining and then confirmed in thin sections after en bloc staining with ruthenium red and potassium permanganate. The previously observed ‘smearing’ of scrapie infectivity over broad ranges in sucrose density gradients may now be explained since these particles were often found as aggregates or chains of particles. The significance of these particles in scrapie infectivity remains uncertain at this time, but absence of these structures in normal tissue fractions may provide a promising new morphological approach to the purification of the infective scrapie agent.

Mild detergent treatment (0.1% Sarkosyl-0.1% β-mercaptoethanol) of Dane particle-rich fraction from human serum resulted in the release of core particles together with HBe antigen activity when examined by the reversed passive haemagglutination method. Furthermore, when the core particles isolated by the above procedure were exposed to stronger detergent (1% Sarkosyl-0.1% β-mercaptoethanol), additional HBe antigen activity was released only from intact core particles with DNA polymerase activity and not from empty core particles.

The maturation of feline syncytium-forming virus (FSFV), a member of the foamy virus sub-family (Spumavirinae), has been studied by electron microscopy of thin sections of infected feline embryo (FEA) cells. The initial event observed was formation of crescent-shaped nucleoids at the plasma membrane. As budding progressed, the nucleoid became circular in outline with an electron-lucent centre in fully mature extracellular particles. These observations suggested that the maturation of FSFV in fully permissive FEA cells resembled that of C-type RNA tumour viruses, rather than the B-type mouse mammary tumour virus. In this respect FSFV may be distinct from other foamy viruses. However, like other foamy viruses FSFV possessed reverse transcriptase activity. Polymerase activity co-sedimented with infectivity in an equilibrium density gradient and exhibited a preference for poly(rA).oligo(dT)10 over poly(dA).oligo(dT)10 as exogenous template.

The effects of human leukocyte interferon (Le-IF) and fibroblast interferon (F-IF) on the growth of the transformed human cell lines, RSa and RSb, were compared. Both were considerably more sensitive to F-IF than to Le-IF. Two cell lines, IFr and F-IFr, derived from the RSa cell line, were resistant to the anticellular effects of Le-IF, but less resistant to those of F-IF.