- Volume 43, Issue 3, 1979

This paper reports the development of a quantal transformation assay for bovine papilloma virus. Support for its specificity to fibropapilloma derived bovine papilloma virus comes from (1) the absence of transformation associated changes following inoculations of normal bovine skin, bovine teat papilloma, bovine teat focal epithelial hyperplasia lesion and canine papilloma derived suspensions; (2) in vitro transformation occurs when CsCl purified fibropapilloma-derived BPV is used; (3) in vitro transformation experiments reported here parallel in vivo transmission of fibropapillomas described elsewhere using the same extracts; (4) the time-temperature inactivation of BPV using in vitro transformation parallels in vivo results and is similar to that reported for other papovaviruses; (5) BPV in vitro transformed cells are tumorigenic in nude mice and increase in vivo resistance in calves to subsequent challenge with fibropapilloma derived virus; (6) inhibition of in vitro transformation by bovine papilloma virus occurs when sera from calves bearing regressing fibropapillomas are used in conjunction with complement; (7) transformation inhibition activity may be adsorbed from high titre sera using BPV-induced tumour cells or in vitro transformed cells but not using various virus suspensions. The detection of BPV in commercially available milk is reported. While it is highly likely that milk-derived BPV is active, no direct evidence is available since the vast majority of teat lesions contain papilloma and focal epithelial hyperplasia-derived BPV which neither transform cultures in vitro, nor produce fibropapillomas in vivo. The work reported here lends further support for the existence of several types of BPV suggested elsewhere.

An enveloped, ether-sensitive, acid-labile virus isolated from Lepomis macrochirus, the bluegill, is described. Virus replication is limited to teleostean cell lines and achieves highest titres in centrarchid cells. As determined by the uptake of tritiated nucleotides and the effect of 6-azauridine and actinomycin D on replication, the virus genome is RNA. The virus has no haemagglutinin or neuraminidase nor does it share antigens with prototypes of orthomyxoviruses, paramyxoviruses or arenaviruses. Thin-section electron microscopy reveals a virion 80 to 100 nm in diam. associated with the cell surface and intracellular vacuoles. The virus appears to mature by budding and exhibits a prolonged replication cycle and significant cell-associated infectivity. The time sequence of replication indicates that the prolonged cycle is due to maturation rather than to delayed synthesis of RNA and protein. The bluegill virus (BGV) represents a novel, hitherto undescribed, type of teleostean virus.

Interaction between phage P22 and phenol-water extracted lipopolysaccharides from sensitive Salmonella bacteria belonging to serogroups A, B and D1 results in hydrolysis of the α-l-rhamnosyl linkages within the tetrasaccharide repeating unit of the O-antigenic polysaccharide chain. These O-antigens have identical structures except for the nature of the 3,6-dideoxy-hexosyl group linked to O-3 of the d-mannosyl residue. Removal of the dideoxysugar, or periodate oxidation followed by borohydride reduction of the l-rhamnosyl residue made the O chain resistant to the endo-rhamnosidase. Substitution of the d-galactosyl residue at O-4, but not at O-6, with an α-d-glucosyl group was compatible with hydrolysis. A number of Klebsiella pneumoniae and Shigella flexneri lipo- or capsular polysaccharides containing chain l-rhamnosyl residues were tested but none was sensitive to the P22 endo-rhamnosidase. The substrate specificity of the endo-rhamnosidase parallels the lytic specificity of the phage which suggests that the initial step in phage P22 infection is a P22 tail enzyme O-antigen substrate interaction. The main product of the hydrolysate was octa-, dodeca- and hexadecasaccharides. Treatment of phage FO resistant smooth strains of S. typhimurium with P22 tails removed O polysaccharide chains and made previously ‘hidden’ FO receptors accessible to the phage.

A sensitive, quantitative and reproducible plaque assay for the measurement of the simian rotavirus SA11 is described. Plaque formation required the presence of the facilitators pancreatin or trypsin and diethylaminoethyl-dextran in the agar overlay. SA11 produced plaques in three continuous primate cell lines: MA-104, CV-1 and LLC-MK2. MA-104 cells were the most sensitive.

Interferons were stimulated in mouse L cells by Newcastle disease virus (NDV) or by polyriboinosinic-polyribocytidylic acid poly(rI). poly(rC). These were fractionated by sequential affinity chromatography on bovine plasma albumin (BPA)-Sepharose and on ω-carboxypentyl (CH)-Sepharose. Based on their interaction with CH-Sepharose, interferor induced by NDV was resolved into three major bands of activity (L/NDV-1,2,3) and poly(rI). Poly(rC)-interferon into two (L/rI:rC-1,2). These interferon components were purified to a specific activity of 3 × 107 to 4 × 107 units/mg protein by antibody affinity chromatography and examined by electrophoresis in SDS-polyacrylamide gels. A total of five molecular species was thus identified for NDV-induced interferon and three for poly(rI). poly(rC) induced interferon, as summarized in Table 1. We conclude from our observations that mouse interferons can be produced by L cells in multiple forms with specific physiochemical properties and in proportions determined by the type of agent employed for induction.

The properties of two temperature-sensitive mutants ts 18 and ts 19 of adenovirus type 5 were studied. It was demonstrated that they had a defect such that they failed to assemble virus and showed defective processing of infected cell polypeptides at the restrictive temperature. Analysis, after protease digestion, of the virions produced at the permissive temperature by SDS PAGE, and of the substrate availability of the mutants to the virus protein kinase suggested that polypeptide VI was defective in these mutants.

A herpesvirus was isolated from a lymphosarcoma culture of tree shrews (Tupaia belangeri) and termed Tupaia herpesvirus 2 (THV-2). Electron microscopy of THV-2 revealed the presence of virus particles with nucleocapsids of about 100 nm surrounded by large envelopes compatible with virions of the herpesvirus group. An extensive host range study revealed the Tupaia embryonic fibroblasts are the cells of choice for the efficient propagation of THV-2. This cell line was used for the continued propagation and plaque assay of THV-2. The mol. wt. of the virus DNA was found to be 100 × 106. The buoyant density of THV-2 was 1.724 g/ml. The DNA of THV-2 was compared to the DNA of herpes simplex virus (HSV) and to another previously isolated herpesvirus from apparently healthy tree shrews (THV-1). The analysis was carried out using the restriction endonuclease EcoRI and the cleavage pattern of THV-2 DNA resulted in DNA fragments which were different from those of HSV-1 DNA and distinguishable from the DNA of THV-1.

When L929 cells were treated with interferon and subsequently with poly(rI). poly(rC), there was a pronounced toxic effect. Most of the cells lysed, but some survived and grew at the same rate as control cells to yield cells which were as sensitive to the effects of interferon and poly(rI). poly(rC) as the original population. The proportion of surviving cells did not vary with either the cell cycle or the cell density. The treated cells produced interferon and some of the interferon was produced by the resistant cells.

Cells which had been X-irradiated before treatment with interferon and poly(rI).poly(rC) behaved similarly so cell division was not necessary for the development of toxicity. The toxic effect also developed when cells were enucleated with the aid of cytochalas in B after treatment with interferon, but not if they were enucleated before treatment. It is concluded that the nucleus is essential for interferon to exert its effect on the cells, but not for the development of cytotoxicity after the addition of poly(rI).poly(rC).

We have tested two possible explanations for the toxic effects observed in L cells treated with interferon followed by poly(rI).poly(rC), namely (1) that the toxicity was preceded by and due to a change in membrane permeability which could be monitored by measurement of ion fluxes, or (2) that the toxicity was due to the action of the inhibitors of protein synthesis which are known to be formed when extracts from interferon-treated cells are treated with poly(rI).poly(rC) in vitro. Neither explanation was found to be correct.

A minor fraction of single-stranded DNA (ssDNA) was isolated by an improved method of hydroxylapatite chromatography (HAC) from the native nuclear DNA (nDNA) of SV-3T3 cells, non-productively transformed by SV40. Molecular hybridization, monitored by the use of S1 nuclease, HAC, isopycnic centrifugation and thermal melting showed that ssDNA from SV-3T3 cells (which amounts to 1.5 to 2% of the total nDNA) has the same characteristics as ssDNA previously isolated from other cell species. Only 27 to 28% of ssDNA can be self-hybridized but the greatest part can be reassociated to the non-repetitive portion of nDNA and up to 38% hybridized to homologous RNAs, as compared with 7 to 8% for bulk nDNA. Highly radioactive virus probes (SV40-3H-cRNA synthesized in a cell-free system and the separated ‘early’ and ‘late’ strands of SV40 DNA labelled with 125I) were annealed to different excess amounts of cellular DNA. Both the quantities of each probe hybridized at saturation levels and the various reaction kinetics indicated that ssDNA is greatly enriched for virus sequences, mainly originating from the ‘early’ DNA strand which is predominantly expressed in SV-3T3 cells. The mode of formation of ssDNA is discussed in the light of other findings on the effects of DNA untwisting proteins and susceptibility of active animal genes to selective enzymic attacks.

The structure of the purified lipid-containing phage PR4 was studied electron microscopically using thin sectioning, negative staining and freeze-fracturing techniques. The lipid layer was located inside a rigid capsid and had a bilayer structure. The innermost dense core was probably composed solely of DNA since no major structural proteins were missing in empty phage particles lacking DNA. During infection, phages were attached to the cell wall of the host. They apparently inject their DNA through the cell envelope. The lipid layer of the phage might play an active role in the injection process. The maturation of the phage capsid takes place within the nuclear region of the cell, whereas intact phages with DNA were always seen in the cell periphery.

Purified preparations of Semliki Forest (SFV) and Sindbis virus haemolyse red blood cells from several species of animals and birds. The optimal haemolysis by SFV was obtained at pH 5.8 with 1-day-old chick erythrocytes incubated at room temperature. Considerable variation in haemolytic activity was observed between different virus preparations purified by different methods. The haemolytic activity of SFV was inhibited by antisera against whole virus or isolated envelope proteins but not with antiserum against virus capsid protein. Neither lipid and detergent-free envelope protein octamers with high haemagglutinating titre, nor isolated nucleocapsids caused haemolysis. Fresh, unpurified SFV and Sindbis virus preparations did not haemolyse unless they were exposed for repeated cycles of freezing and thawing. It appears that the haemolytic activity resides in the virus glycoproteins(s) but can only be manifested in slightly damaged whole virus particles.

RNA extracted from particles of five nepoviruses [raspberry ringspot (RRV), strawberry latent ringspot, tobacco ringspot (TRSV), tomato black ring (TBRV) and tomato ringspot viruses] was bound to oligo(dT)-cellulose in buffers of high ionic strength (HS) whereas RNA from particles of tobacco mosaic virus or tobacco rattle virus was not. This suggests that nepovirus RNA molecules contain polyadenylate [poly(A)]. At least 97% of the infective RNA molecules of TRSV and TBRV were bound in HS buffer and eluted in buffer of low ionic strength. The two species of genome RNA of RRV and TBRV were bound equally to oligo(dT)-cellulose, but the satellite RNA (RNA-3) of TBRV was bound less avidly and to a smaller extent than the genome RNA.

When 3H-borohydride-labelled TRSV RNA was digested with ribonucleases A + T1 about 29% of the radioactivity bound to oligo(dT)-cellulose, presumably as polyadenylate. Adenosine trialcohol was the only nucleoside trialcohol detected in alkali digests of borohydride-labelled RNA. Thus polyadenylate is probably located at the 3′-termini of the RNA molecules.

Total nucleic acids prepared from a number of murine retroviruses have been shown to contain virus-specific DNA in addition to genomic RNA. This virus-specific DNA has been shown to be at least partially double stranded and to be present within the virus core particle. The DNA isolated from the virus is greatly enriched in virus-specific DNA relative to that from virus infected cells.

The morphology of the virions and nucleocapsids of paramyxovirus 107 (PMV 107) and the replication of the virus were investigated by electron microscopy. The virions and nucleocapsids exhibited the same structural properties as other paramyxoviruses. Nucleocapsids were found in the nucleus and cytoplasm of infected bovine embryonic lung (BEL) cell cultures. A similar situation has been described for the morbilliviruses measles, SSPE, distemper and rinderpest. Alignment of nucleocapsids beneath the plasma membrane and budding of PMV 107 in the productive BEL cell infections were also similar to the morbillivirus-infected cells. In a line of monkey cells (CV1) persistently infected with PMV 107 only cytoplasmic nucleocapsids could be demonstrated. On the basis of its morphology and morphogenesis it is suggested that PMV 107 should be classified as a paramyxovirus. Since nucleocapsids could also be found in the nucleus of infected BEL cells the morphogenesis of PMV 107 closely resembles that of viruses of the morbillivirus group.

Measles virus Edmonston strain was purified by ultrafiltration followed by two successive sedimentations through sucrose. Purified virus retained infectivity and, when used as an immunogen, elicited high titred antibody to measles antigens by conventional serology. The measles preparations were examined by SDS-PAGE followed by staining. In addition, following PAGE, the purity of these preparations was assessed immunochemically using antisera directed to measles and host cell antigens. The results of these studies demonstrate the utility of the purification method for the preparation of milligram quantities of relatively pure measles virus.

A virus unrelated to any other from bees was isolated from diseased adults of Apis mellifera from Egypt. It has isometric particles about 30 nm in diam., which contain RNA and sediment at 165S. The particles contain three proteins, have a buoyant density in CsCl of 1.37 g/ml, aggregate readily in low concentrations of buffer or at low pH values, form ‘empty’ shells below pH 5.0 and disintegrate below pH 4.0.

Three virus isolates closely related to each other and to Kashmir bee virus were obtained from dead adults, larvae and prepupae of Apis mellifera from South Australia, New South Wales and Queensland. Particles of the four strains are physically indistinguishable and each contains three proteins, but two proteins of the Australian strains differ from those of Kashmir bee virus in estimated size and stability.

The anomalous viroid-like RNA associated with cadang-cadang disease of coconut palms (ccRNA-1) has been cleaved by treatment with the single-strand specific nuclease S1, polyadenylated and used as a template for the oligo(dT) primed synthesis of complementary DNA (cDNA) by the avian myeloblastosis virus reverse transcriptase.

The efficiency of synthesis was low, with only 3 to 4.5 ng of cDNA synthesized from 2 µg of RNA. Most of the cDNA was in the 4S size class. A R0t½ value of 1 × 10−3 mol. s/l was obtained when this cDNA was hybridized with ccRNA-1, consistent with ccRNA-1 representing a unique species of mol. wt. approx. 100000. The maximum hybridization value obtained with ccRNA-1 was approx. 50%; the S1 nuclease resistance of the cDNA after self-annealing was approx. 7%. The melting behaviour of the homologous hybrids provided evidence for the specificity of base-pairing with no evidence of mismatching.

The cDNA has been shown to be a specific probe for cadang-cadang associated RNA. It has been used to demonstrate that ccRNA-1 and ccRNA-2 have common nucleotide sequences, that ccRNA-1 is uniquely associated with diseased and not healthy palms and that it has no significant homology with high mol. wt. RNA or DNA from diseased palms. The value of the cDNA as a diagnostic probe for ccRNA-1 in crude nucleic acid extracts has been demonstrated.

Human adenovirus (Ad) serotypes provide an early factor(s) that is necessary for adenovirus-associated virus (AAV) multiplication in human cell lines. However, little, if any, AAV production occurs in primary African green monkey kidney (AGMK) cells co-infected with AAV and a helper human Ad (non-permissive infection), unless cells are additionally infected with SV40 (permissive infection). To determine the basis of the host restriction of AAV replication in AGMK cells, AAV DNA, RNA and protein synthesis were analysed under various conditions of infection. Hybridization reactions revealed no detectable AAV-specific DNA or RNA in infections with AAV alone or in combination with SV40. In co-infections with AAV and Ad5 or Ad7, the synthesis of both AAV- and Ad-specific DNA and RNA occurred without a significant rise in titre of either virus. During non-permissive infection, however, AAV DNA synthesis was abnormal in that an expected accumulation of single-stranded progeny molecules was not observed. Finally, although intact 20S AAV transcripts were present in the cytoplasm of AGMK cells during non-permissive infection (in amounts ranging from 50 to 80% of that found during permissive infection), AAV-specific polypeptides were not demonstrable by polyacrylamide gel electrophoresis. Taken together, these experiments indicate that the host restriction of AAV replication in AGMK cells is exerted at the level of translation of the single AAV messenger RNA. In addition, it appears that one or more of the AAV polypeptides specified by this message is required for the production of single-stranded AAV progeny DNA.

The presence of a C-type virus in tissues of an embryonic rhabdomyosarcoma of a corn snake Elaphe guttata was previously described, based upon electron microscopic observations. A virus, corn snake retrovirus (CSRV) has been recovered from the tumour tissue by inoculation of a tissue homogenate on to either the rattlesnake fibroma cell line or early passage cells derived from rattlesnake heart or kidney. Attempts to cultivate the virus in other reptilian cell systems were unsuccessful. The virus was classified as a retrovirus on the basis of electron microscopic observations of fine structure and morphogenesis, and the demonstration of virion-associated reverse transcriptase and a buoyant density of 1.16. Polypeptide analysis of CSRV performed by polyacrylamide gel electrophoresis revealed the presence of five major polypeptides: three had mobility analogous to that of structural polypeptides of viper retrovirus (VRV) but two polypeptides, one of mol. wt. approx. 16000 and a glycoprotein of mol. wt. approx. 72000, were unique. Antigenic comparison of CSRV and VRV by agar gel immunodiffusion revealed that CSRV possesses a major antigenic determinant which is different to that of VRV. CSRV propagated in rattlesnake fibroma cells was demonstrated to be slowly cytopathic for rattlesnake heart and kidney cells in vitro.