- Volume 37, Issue 3, 1977

Rotavirus-infected intestinal epithelial cells in vivo and calf kidney cells in vitro have been examined by electron microscopy. Morphogenesis takes place in the cytoplasm and several particle types are observed. These can be classified broadly into two groups, one of which appears to represent the characteristic normal development of infectious virus and the other, distinctly different, which may be a non-infectious form. Two of the particle types are tentatively identified as the ‘single’ and ‘double’ capsid rotavirus particles seen typically in negatively stained preparations.

A new temperature sensitive mutant of adenovirus type 2 was isolated and characterized. H2ts48 is DNA positive but fails to synthesize stable 95K, 80K and Va (Pre-VI) polypeptides. In addition, many polypeptides exhibit reduced stability during pulse-chase experiments. No virion particles of any type are assembled although a new type of intranuclear core-like structure was observed in thin sections by electron microscopy. Hexon polypeptides (120K) are synthesized as demonstrated by SDS-polyacrylamide gel electrophoresis, but immunofluorescence, immunodiffusion and sucrose velocity gradient analyses show that no hexon capsomere antigens (360K) are assembled. Similarly, fibre polypeptides are synthesized normally, but the immunological and sedimentation properties of fibre are abnormal. Because this mutant failed to complement several complementing adenovirus mutants, and depressed the growth of wild type virus, it was concluded that ts48 may be a novel, trans-dominant mutation.

The tissue culture-adapted P3 clone of MOPC-21 BALB/c myeloma cells produce a C-type virus, designated as MO21-MuMAV, that has many properties in common with the NB-tropic, C-type virus produced by a clone of the FLOPC-1 line of BALB/c myeloma (FL1-MuMAV) and one property that is distinctly different. On the one hand, both particles are extremely unstable as shown by their buoyant densities when analysed under different conditions of isopycnic gradient centrifugation; the enzymic activity of the virus RNA-dependent DNA polymerase is similar; the two viruses are similar antigenically; both viruses have similar XC syncytium-inducing activity; the endogenous RNA of both viruses contains poly(A) tracts of a similar length; both are NB-tropic as they productively infected BALB/3T3 and NIH/3T3 cells. However, the RNA of MO21-MuMAV consists of more high mol. wt. (60 to 70S) RNA species than does FL1-MuMAV RNA. In addition, MO21-MuMAV does not infect normal rat kidney cells as efficiently as does FL1-MuMAV. The NB-tropism of MO21-MuMAV does not appear to be due to a mixture of N- and B-tropic viruses on the basis of (a) the NB-tropism of the progeny virus produced by infected NIH/3T3 and BALB/3T3 cells; and (b) the dose-response relationship of MO21-MuMAV infection of BALB/3T3 and NIH/3T3 cells. Thus, the data suggest that different BALB/c myelomas produce C-type viruses that are very similar, but not necessarily identical.

Haemagglutinating encephalomyelitis virus (HEV), a member of the coronavirus group, was adapted for growth in adult pig thyroid cell cultures and purified by ammonium sulphate precipitation and rate zonal centrifugation through sucrose gradients. Polyacrylamide gel electrophoresis of samples of purified virus revealed the presence of five polypeptides, four of which contained carbohydrate. The molecular weights of these proteins were 180000 (gp 180), 125000 (gp 125), 100000 (gp 100), 56000 (p 56) and 26 500 (gp 26.5). After treatment of the virus with the non-ionic detergent Nonidet P40, two subviral components were isolated. As RNA-containing particle, sedimenting in sucrose gradients at the same rate as untreated virus, was analysed and found to contain two polypeptides, p 56 and gp 26.5. The second complex sedimented at a much slower rate and contained three glycoproteins gp 180, gp 125 and gp 100. Comparison of these findings with data published for other members of the coronavirus group is discussed.

Mol. wt. estimates have been derived for the covalently-closed circular DNAs extracted from seven nuclear polyhedrosis and three granulosis viruses by a comparison of their contour length with that of the replicative form of f1 bacteriophage DNA. The length of the circular DNA molecules was uniform for each of the ten viruses studied. The mol. wt. values obtained ranged from 58 × 106 to 94 × 106 although all but two values were within the range 69 × 106 to 81 × 106. There appeared to be two major size classes within the latter range. The mol. wt. of the three granulosis virus DNAs were from 68 × 106 to 71 × 106 while the nuclear polyhedrosis virus DNAs spanned the full range. Thus, the distribution of mol. wt. values obtained from covalently-closed DNAs isolated from baculoviruses was not consistent with the division of the group into nuclear polyhedrosis and granulosis viruses.

Intracytoplasmic A particles can be found in the cytoplasm of mammary tumours, certain leukaemias and Leydig cell tumours of the mouse. These intracytoplasmic A particles share antigenic determinants with the mouse mammary tumour virus (MTV) and are considered to be precursors of MTV B-type particles on the basis of morphological comparison. Both DNA and RNA have been reported to be associated with these intracytoplasmic A particles (Tanaka, Tamura & Tsujimura, 1972; Smith, Litwach & Longfellow, 1974). In this study it is shown that (1) the RNA of intracytoplasmic A particles is homologous to the RNA of MTV; (2) the DNA of the intracytoplasmic A particles is not a replicative form of the MTV genome, because no hybridization was observed between this intracytoplasmic A particle DNA and the radioactively labelled RNA or cDNA of MTV; (3) the intracytoplasmic A particles also contain an RNA-directed DNA polymerase activity with a cation preference which is similar to that of the reverse transcriptase of the MTV in a poly(rC).oligo(dG)-directed DNA polymerase assay.

Ly cells were treated with 100 reference units/ml of mouse interferon and then infected with a wild-type vesicular stomatitis virus (VSV) at a multiplicity of 10 to 60 p.f.u./cell. Prolonged infection of cultures ensued, lasting from 14 to at least 60 days. Less than 1% of the cells produced infectious virus, but more than 10% produced detectable levels of VSV antigens. No small virion RNA forms (< 42S) characteristic of defective interfering (DI) virus particles were detected. The virus produced appeared to be temperature sensitive. There was decreased plaquing efficiency at 37 or 39 °C compared to 32 °C and termination of the chronic infection due to c.p.e. within a few days after shift to 32 °C. The cultures resisted superinfection with wild-type VSV or with a heterologous virus, encephalomyocarditis (EMC) virus. Treatment of cultures with rabbit anti-mouse interferon globulin resulted in a marked increase in virus titres and termination of the chronic infection. Prolonged VSV infection in this system may be related both to the emergence of temperature-sensitive mutants and to endogenous interferon production rather than to cyclical generation of DI particles.

Extracts from mouse interferon-treated mouse cells (Ehrlich ascites tumour and L929) were reported earlier to differ in several characteristics from extracts of untreated cells. We report now that the effect of treatment with human interferon of cells of a human line (HeLa S3) is similarly manifested in the cell extract. A comparison of extracts from interferon-treated HeLa S3 cells (S30 int ) with that from untreated cells (S30c) revealed: (a) an impairment in S30 int of the methylation of (unmethylated) reovirus mRNAs by the cellular and virus enzymes; (b) a faster degradation of reovirus mRNAs in S30 int but only in reaction mixtures supplemented with double-stranded RNA and ATP; (c) an increased phosphorylation by ATP of one (or two) proteins in S30 int but only in reaction mixtures supplemented with double-stranded RNA and (d) a more pronounced inhibition of translation by double-stranded RNA in S30 int . No such effects were observed in extracts prepared from HeLa cells treated with mouse interferon.

Neuraminidase activity could be demonstrated in highly concentrated preparations of human parainfluenza 1 virus, strain C 35 (HA2 virus). Among the substrates used, the most suitable are N-acetyl neuramin lactose and fetuin. Mucin type I and type II were not hydrolysed. The neuraminidase exhibited some characteristics similar to those of the other paramyxoviruses (Sendai, NDV, mumps, human parainfluenza 2 virus): the optimum pH ranged between 5 and 5.4, and the K m value was 5 × 10−3 m when tested with N-acetyl neuramin lactose. Its optimum activity was between 37 and 40 °C and it was thermolabile, the enzymic activity being reduced to 50% in 5 min at 45 °C and entirely destroyed at 50 °C in the same period. The thermal inactivation constants of neuraminidase and haemagglutinin and the temperature which inactivated 50% of both these activities were very similar to those already shown for NDV. Haemagglutinin and neuraminidase activities were rapidly destroyed by ionic detergents, but not by non-ionic detergents.

Vesicular stomatitis virus (VSV) in mixed infection with Sindbis virus (SbV) produces a proportion of phenotypically mixed particles (pseudotypes) containing VSV genomes and neutralization antigen(s) provided by SbV. This was demonstrated by heat-stabilization of the thermolabile tlB17 mutant of VSV and by neutralization with corresponding antisera. Phenotypic mixing is apparently unilateral because no SbV(VSV) pseudotypes could be found. Similarly, avian RNA tumour virus (ATV) in mixed infection with Sindbis virus produces a proportion of phenotypically mixed particles containing ATV genomes and SbV antigens, but no detectable particles containing SbV genomes and ATV envelope antigens. SbV acts as a helper virus for envelope-defective Rous sarcoma virus (RSV). When an avian helper virus is also present in the mixed infection, more than 90% of the RSV particles bearing SbV envelope antigens also bear ATV envelope antigens and are doubly neutralizable by antisera specific to either parent virus. In mixed infection of Langat virus and VSV, a proportion of doubly neutralizable particles containing VSV genomes were produced, but no pure pseudotypes. These results indicate that in mixed infections between enveloped animal viruses, VSV and ATV readily assemble foreign envelope glycoproteins, but that SbV does not. In certain phenotypically mixed virus stocks, only doubly neutralizable particles are found and are presumed to bear a mosaic of envelope antigens; in other stocks, particles can also be detected which are resistant to neutralization by antiserum specific to the envelope antigen encoded by their genomes, and these are presumed to represent pure pseudotypes.

Treatment of a fully permissive monkey kidney cell clone (CV1Cl1) with 5-iodo-2′-deoxyuridine (IdUrd) before infection with SV40 virus enhances the yield of virions 10- to 50-fold. The increase is detectable only after slowing down the virus growth cycle by reducing the m.o.i. and by incubating at low temperature. The IdUrd pre-treatment enhances SV40 DNA replication and the number of V-antigen and virus-synthesizing cells. The potentiating effect of IdUrd is not observed when the pre-treated cells are infected with SV40 DNA. The synthesis of SV40 T-antigen is increased even in the presence of cytosine arabinoside (Ara-C). IdUrd inhibits cellular DNA synthesis but the incorporation of 3H-uridine and 3H-leucine into RNA and proteins is not affected. Late virus functions are preferentially expressed in the cells in which cellular DNA synthesis is inhibited. The results suggest that the enhancement by IdUrd of SV40 replication would be the consequence of at least two complementary events: (1) stimulation of an early virus function localized between the arrival of the virus DNA in the nucleus and T-antigen induction; (2) inhibition of cellular DNA synthesis with a consequent greater availability of cellular factors required for virus growth.

Capsid types present in hepatocyte nuclei of Syrian hamsters infected with the animal strain of equine herpesvirus type 1 (EHV-1H) were studied and characterized. Three capsid species were isolated: L capsids (ρ = 1.23 g/ml) which are devoid of a core structure and appear to be empty shells, I capsids (ρ = 1.24 g/ml) which contain an electron-lucent, cross-shaped, immature core, and H capsids (ρ = 1.25 g/ml) which contain an electron-dense, mature core structure. All three capsids first appeared at approx. 6 h post inoculation and were present in a ratio of approx. 10:87:3 (L:I:H) at all times during infection. Analysis of the polypeptide and amino acid compositions of certain species indicated that these capsids are identical to L, I and H capsids isolated from cell cultures infected with the culture tissue strain of EHV-1. These findings support the model (O’Callaghan & Randall, 1976) that I capsids are a major precursor of mature capsids and play a major role in the maturation of this herpesvirus.

Duvenhage virus was originally isolated in South Africa from the brain of a man who had been bitten by a bat and died after a rabies-like illness. Previous immunofluorescence tests indicated that the virus was distinct from rabies virus. In the present study an antigenic relationship of this virus to rabies is defined, and pathological, morphological and further serological characterization is presented. Duvenhage virus-infected mice developed a central nervous system disease characterized by a short incubation period, a moderate degree of inflammatory infiltration of brain parenchyma and by small intraneuronal inclusion bodies. By electron microscopy typical rhabdovirus particles were found budding upon endoplasmic reticulum and plasma membranes of brain neurons. In these characteristics Duvenhage virus resembled laboratory or ‘fixed’ strains of rabies virus. The structural polypeptide composition of Duvenhage virus was very similar to that of rabies virus. Duvenhage virus could be distinguished from rabies by in vivo neutralization and cross-challenge tests in mice, and to a lesser extent by complement-fixation and fluorescent antibody tests. Antibody to purified ribonucleoproteins used in indirect immunofluorescence tests did not distinguish between rabies and Duvenhage virus. In vitro neutralization tests using antisera against whole virus, and against purified virus glycoprotein, confirmed the distinction; consequently Duvenhage virus should be considered a new member of the rabies serogroup (i.e. Lyssavirus genus, Rhabdoviridae family).

The native and denatured 60 to 70S RNAs of two murine oncornaviruses and one simian oncornavirus were examined for their poly (A) content. In the native genome, a significant proportion of poly (A)− RNA was found and intact 60 to 70S RNA complex was identified in this fraction. The amount of poly (A)− RNA in the native genome was related to cellular growth and seemed to be independent of virus maturation. The subunits obtained after thermal denaturation consisted of approx. 2/3 poly (A)+ and 1/3 poly (A)− RNA.

The poly (A)− subunits were mainly composed of 20 to 28S RNA and the poly (A)+ subunits of 30 to 35S RNA. The results of competitive molecular hybridization of these two fractions with virus cDNA suggested that the two species of subunits possessed similar nucleotide sequences.

Various double-stranded RNAs of either synthetic or natural origin have been compared for their interferon-inducing potency in human skin fibroblasts ‘primed’ with interferon and ‘superinduced’ with cycloheximide and actinomycin D. While natural double-stranded RNAs (extracted from either Penicillium chrysogenum mycophage, f2 bacteriophage or reovirus) and alternating copolymers [(A-U)n.(A-U)n, (G-C)n.(G-C)n, (I-C)n.(I-C)n]* proved relatively less effective in inducing interferon than (I)n.(C)n, a variety of synthetic homopolymer pairs, including (I)n.(br5C)n, (I)n.(s2C)n, (A)n.(rT)n and (A)n.(U)n, showed an interferon-inducing activity comparable to that of (I)n.(C)n.

The neuraminidase of B/Lee/40 influenza virus was isolated following detergent dissociation or tryptic digestion of virus particles and the amino acid and carbohydrate compositions of the two preparations are reported.

The results indicate that the carbohydrate side-chains of the neuraminidase contain only N-acetylglucosamine, galactose, mannose and fucose, that they are attached by N-acetylglucosamine-asparagine linkages, and that the molecular weights of the neuraminidase subunit and the intact molecule are about 70000 and 280000, respectively. The results also suggest that more than 50% of the carbohydrate is attached to the membrane-associated ‘stalk’ of the molecule and that in this ‘stalk’ region the subunits are linked by disulphide bonds.

Cultures of human fibroblasts trisomic for chromosome 21 were more sensitive to human leukocyte and human fibroblast interferons than were human diploid fibroblasts; these in turn were more sensitive than fibroblasts monosomic for chromosome 21. The sensitivities of these cells to human interferons correlated with the amounts of interferons which they bound. These data indicate that some gene on chromosome 21 codes for an interferon-receptor component.

Also, interferon from a heterologous species (mouse) was respectively much more and somewhat more active in trisomic and disomic cells than in monosomic cells. These data suggest that heterologous and homologous interferons may share common receptor component(s) coded by chromosome 21. Alternatively, human chromosome 21 may carry genetic information for some factor(s) responsible for increasing the exposure of certain surface receptors which are themselves coded by other chromosomes.

Subcutaneous injection of microgram quantities of purified SV40 DNA into newborn Syrian hamsters induced sarcomas. The tumour cells contained SV40 T-antigen and virus could be rescued from the cultured tumour cells. Addition of poly-L-ornithine or calf thymus DNA to the injection mixture did not enhance the tumour incidence. SV40 DNA cleaved with endonuclease EcoRI also displayed oncogenic potential.

Chick embryos of two test-crosses, involving three inbred lines, 7-2, E and Reaseheath C, of Houghton Poultry Research Station, were challenged (on to the chorioallantoic membranes) with RSV(RAV 2) of subgroup B and RSV(RAV 0) of subgroup E. Genetic analysis for B-and E-susceptibility indicated that the average E-susceptibility in the chicken line E carrying the e s gene can be increased or decreased by addition or subtraction, respectively, of the b s gene. The results of this study are a better fit to a two-gene model with complementary gene action than to a single gene model of multiple allelism of the tvb-locus with pleiotropic gene action.